Botulinum toxin cell binding domain polypeptides and methods of use for treatments of fibrosis associated disorders

ABSTRACT

A polypeptide having an amino acid sequence corresponding to a binding domain of a botulinum toxin is described. The polypeptide modulates expression of genes involved in, for example, collagen production and extra cellular matrix organization, and finds use, therefore in treating or reducing the occurrence of a disease such as a disease of the ECM, a connective or epithelial tissue disease, an endothelial disease or a fibrotic disease, e.g., a fibrotic disease of the ECM, connective tissue, or endothelium. Nucleic acids encoding the polypeptide, as well as vectors, host cells, and systems comprising the nucleic acids, are further described.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional patent application of U.S. patent application Ser. No. 16/228,285, filed Dec. 20, 2018, which claims the benefit of U.S. Prov. No. 62/608,119, filed on Dec. 20, 2017 and U.S. Prov. No. 62/727,640, filed on Sep. 6, 2018, the entire contents of which are incorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Dec. 19, 2018, is named 19980-US-A-NTB_SL.txt and is 71,997 bytes in size.

TECHNICAL FIELD

The present disclosure relates generally to polypeptides from the cell binding domain of Botulinum toxin and use of the polypeptides for therapeutic applications, including diseases and conditions associated with fibrosis.

BACKGROUND

The anaerobic, gram positive bacterium Clostridium botulinum produces a potent polypeptide neurotoxin, botulinum toxin (BoNT), which has well-documented medical applications. Naturally-occurring Clostridial toxins are each translated as a single-chain polypeptide of approximately 150 kilo Daltons (kDa) of an approximately 50 kDa light chain (LC), comprising an enzymatic domain, and an approximately 100 kDa heavy chain, comprising an N-terminal translocation domain (H_(N)) and a C-terminal receptor-binding domain (H_(C)). Thus, full length Clostridial toxin molecules comprise three functionally distinct domains: (1) an enzymatic domain located in the LC that includes a metalloprotease region containing a zinc-dependent endopeptidase, which specifically targets core components of the neurotransmitter release apparatus; (2) a translocation domain contained within the amino-terminal half of the heavy chain (H_(N)) that facilitates release of the LC from intracellular vesicles into the cytoplasm of the target cell; and (3) a binding domain found within the carboxyl-terminal half of the heavy chain (H_(C)) that determines the binding affinity and specificity of the toxin to receptors located at the surface of the target cell. The binding domain comprises two distinct structural features of roughly equal size designated the N-terminal subdomain (H_(CN)) and the C-terminal subdomain (H_(CC)).

This toxin architecture is present in various immunologically-distinct botulinum neurotoxins, e.g., botulinum neurotoxin serotypes A, B, C₁, D, DC, E, F, G, X and J. Regardless of serotype, there exists a remarkable degree of structural and functional homology between the full-length 150 kDa toxin proteins as well as the individual domains therein. In this regard, the BoNTs possess approximately 35% amino acid identity with each other and share the same functional domain organization and overall structural architecture. Within each type of Clostridial toxin there are also subtypes that differ somewhat in their amino acid sequence; however, the domain architecture of the various domains, e.g., endopeptidase, translocation, and the cell-binding domains, are also conserved within the individual subtypes. For example, there are presently five BoNT/A subtypes, BoNT/A1, BoNT/A2, BoNT/A3 BoNT/A4 and BoNT/A5; which share approximately 89% overall amino acid identity. Other members of the superfamily, e.g., tetanus toxin (TeNT) produced by a uniform group of C. tetani and other related toxins produced by Clostridia species, e.g., BaNT (produced by C. baratii) and BuNT (produced by and C. butyricum), are also structurally similar to the aforementioned Clostridial toxins, e.g., BoNT/F and BoNT/E, for instance, with respect to amino acid sequence identity.

Existing pharmaceutical formulations contain the full-length Clostridial toxin. There is a need for Clostridial toxin fragments and variants which have biological activities that are comparable to, if not better than, full length, while offering an improved therapeutic profile, e.g., improved safety, enhanced stability and/or better in vivo efficacy, compared to the whole Clostridial toxin.

SUMMARY

Described herein are various therapeutic applications of BoNT polypeptides of the present disclosure. For instance, treatment with a polypeptide containing the binding domain of BoNT/A (H_(C)/A), fragments or variants thereof modulated the expression of a number of genes in normal human primary fibroblasts. For example, treatment of normal human primary fibroblasts with with the polypeptides of the present disclosure resulted in up-regulation of matrix metalloproteinases MMP1 and MMP3, and metalloproteinase inhibitor TIMP1, which are involved with extracellular matrix (ECM) breakdown and re-modeling, and down-regulation of S100 calcium-binding protein A4 (S100A4), a known marker of fibrosis and inducer of alpha-smooth muscle actin(α-SMA), and down regulation of α-SMA/ACTA2 (Actin, Alpha 2, Smooth Muscle, Aorta), known to be associated with fibroblast contractility and myofibroblast formation. These observations suggest that polypeptides of the disclosure affect expression of proteins involved with creating, maintaining and repairing the dermal Extra Cellular Matrix (ECM), for example by removing excess or damaged ECM components. Since formation of scars and fibrosis is associated with aberrant production of ECM components, the data suggest that polypeptides of the disclosure have anti-scarring and anti-fibrotic effects. Treatment with the binding domain of BoNT/A (H_(C)/A) also resulted in significant dose-dependent changes in expression of 18 fibroblast genes based on qPCR. Notably, expression of genes known to be involved with tissue and ECM homeostasis, re-modeling, renewal, and repair were increased, while expression of the two genes associated with fibrosis was decreased.

Additionally, treatment with the polypeptide of the disclosure reduced secretion of procollagen peptides, which are precursors of mature collagen fibers, from primary dermal scar derived fibroblasts, suggesting that the polypeptide affects production of collagen, which is the primary component of the dermal Extra Cellular Matrix (ECM). Since formation of scars and fibrosis is associated with aberrant production of collagen, these observations suggest that polypeptides corresponding substantially to the binding domain of BoNT/A (H_(C)/A) have anti-scarring and anti-fibrotic effects. The effect is similar to or more than, the effect of anti-CTGF and anti-TGFβ antibodies, two known anti-fibrotic agents, in this study, suggesting that polypeptides of the disclosure have similar or additional anti-fibrotic effects.

Treatment with the polypeptides of the disclosure, e.g., BoNT/A (H_(C)/A), reduced contractility of primary dermal keloid scar-derived fibroblasts in a fibrin hydrogel assay. Fibroblast contractility is associated with fibroblast to myofibroblast transition and scar formation, for example as the fibroblasts contract to close a wound or transform an existing scar. The observed data therefore suggest that polypeptides corresponding substantially to the binding domain of BoNT/A (H_(C)/A) could inhibit contractility and reduce scar formation during wound closure in patients.

The polypeptides of the disclosure, e.g., BoNT/A (H_(C)/A), affected secretion of a number of proteins from fibrotic epithelial human breast cancer cells, where fibrosis is induced by hypoxia, e.g., reduced secretion of fibronectin, collagen IV, and thrombospondin-1, key structural components of the dermal ECM, and increased secretion of MMP1 and TIMP1, metalloproteinase and metalloproteinase inhibitor proteins that are involved with creating, maintaining and repairing the dermal ECM, for example by degrading excess or damaged ECM components. Since formation of scars and fibrosis is associated with aberrant production of collagen and other ECM components, these observations suggest that polypeptides of the disclosure or fragments thereof, e.g., N-terminal half (H_(CN)), have anti-scarring and anti-fibrotic effects. The effect is similar to, or more than, the effect of anti-CTGF (FG-3019) antibody, a known anti-fibrotic agent. The data point to use of the polypeptides of the disclosure, e.g., BoNT/A (H_(C)/A), or fragments thereof, e.g., BoNT/A (H_(CN)/A), in reducing hypoxia induced fibrosis, e.g., fibrousness of tissues and organs, and scars in human patients, resulting in for example inhibition or reversal of tissue and organ fibrosis and reduction of visual scars. Particularly, in experiments with keloid fibroblasts, the data show that polypeptides of the disclosure, e.g., BoNT/A (H_(C)/A), or fragments thereof, e.g., BoNT/A (H_(CN)/A), modulate secretion of fibronectin from human keloid scar fibroblast (KF116R) cells, a key component of the dermal ECM and a mediator in the formation of scars and fibrosis.

Additional studies showed that the polypeptides of the disclosure, e.g., BoNT/A (H_(C)/A), reduced secretion of proteins from normal human primary fibroblasts (HDFa), including collagens and lactotransferrin, the latter of which has been shown to increase fibroblast contractility. Since formation of scars and fibrosis is associated with aberrant production of collagen and increased contractility, this suggests that polypeptides of the disclosure have anti-scarring and anti-fibrotic effects.

Parallel studies with BoNT/DC (H_(C)/DC) showed similar results. For example, H_(C)/DC treatment modulated the expression of a number of genes in human scar derived primary fibroblasts, including up-regulation of MMP1 and MMP3, and metalloproteinase inhibitor TIMP1, which are involved with extracellular matrix breakdown and re-modeling, and down-regulation of connective tissue growth factor (CTGF), a known inducer of fibrosis, and alpha-smooth muscle actin (α-SMA/ACTA2), which is associated with fibroblast contractility and a marker of myofibroblast formation. H_(C)/DC was found to be more effective at modulating some gene expression compared to H_(CN)/A, suggesting that different BoNT serotypes have different effects. These findings suggest that other additional BoNT serotypes could affect fibrosis in a manner that is analogous to BoNT/A (H_(C)/A) and BoNT/DC (H_(C)/DC).

The disclosure relates to the following non-limiting embodiments:

In one aspect, polypeptides and polynucleotide sequences, and compositions comprising such polypeptides or polynucleotides, which comprise a binding domain sequence of a Clostridial toxin, such as botulinum toxin, are described. In one embodiment, polypeptide sequences are described that participate in cellular signaling and/or that modulate cellular gene expression in a target cell to alleviate or reduce the occurrence of a condition or disorder associated with extracellular matrix (ECM) dysregulation. In one embodiment, polypeptide sequences are described that repair the ECM by for example upregulating expression of genes involved in ECM organization, repair, renewal such as for example MMP1 (Matrix metalloproteinase-1), MMP3 (Matrix metalloproteinase-3), TIMP1 (Metallopeptidase inhibitor 1). In another embodiment, the polypeptide sequences alleviate or reduce the occurrence of a fibrotic disorder by for example down-regulating expression and/or secretion of proteins associated with fibroblast contractility, myofibroblast formation and/or scar formation.

In another aspect, there is provided a polypeptide comprising an amino acid sequence substantially identical to an amino acid sequence in a binding domain of a botulinum toxin. In some embodiments, the polypeptide has a molecular weight between about 1 kDa to about 90 kDa. In one embodiment, the molecular weight of the polypeptide is between about 4 kDa to about 60 kD. In one embodiment, the molecular weight of the polypeptide is between about 12 kDa to about 50 kD.

In another aspect, a polypeptide comprises a sequence of amino acids having at least 90% sequence identity to a binding domain of a botulinum toxin is provided. In some embodiments, the polypeptide has a molecular weight of the polypeptide is between about 1 kDa to about 90 kDa. In one embodiment, the molecular weight of the polypeptide is between about 4 kDa to about 60 kD. In one embodiment, the molecular weight of the polypeptide is between about 12 kDa to about 50 kD.

In some embodiments, the polypeptide comprises an amino acid sequence substantially identical to an amino acid sequence in the full-length of a botulinum toxin, which is devoid of toxicity. In one embodiment, the polypeptide comprises an amino acid sequence substantially identical to the amino acid sequence of the full-length of a botulinum toxin which is devoid of toxicity. In some embodiments, the polypeptide comprises an amino acid sequence substantially identical to an amino acid sequence in the heavy chain of the botulinum toxin. In some embodiments, the polypeptide comprises an amino acid sequence substantially identical to an amino acid sequence in the carboxyl or C-terminal segment of the heavy chain of botulinum toxin (H_(C)). In one embodiment, the polypeptide comprises an amino acid sequence substantially identical to an amino acid sequence of the binding domain of the botulinum toxin. In one embodiment, the polypeptide comprises an amino acid sequence identical to the amino acid sequence of the binding domain of the botulinum toxin. In another embodiment, the polypeptide comprises an amino acid sequence substantially identical to an amino acid sequence in the amino or N-terminal half of the binding domain of the botulinum toxin (H_(CN)). In one embodiment, the polypeptide comprises an amino acid sequence identical to the N-terminal half of the binding domain of the botulinum toxin.

In one embodiment, the botulinum toxin is selected from the group consisting of Botulinum toxin serotype A (BoNT/A), Botulinum toxin serotype B (BoNT/B), Botulinum toxin serotype C₁ (BoNT/C₁), Botulinum toxin serotype D (BoNT/D), Botulinum toxin serotype E (BoNT/E), Botulinum toxin serotype F (BoNT/F), Botulinum toxin serotype G (BoNT/G), Botulinum toxin serotype H (BoNT/H), Botulinum toxin serotype X (BoNT/X), Enterococcus sp. Botulinum toxin J (eBoNT/J), and mosaic Botulinum toxins and/or variants thereof. Examples of mosaic toxins include BoNT/DC, BoNT/CD, and BoNT/FA. In one embodiment, the botulinum toxin is not Botulinum toxin serotype A (BoNT/A).

In some embodiments, the polypeptide is capable of modulating expression of fibrosis associated proteins. In one embodiment, the polypeptide is capable or reducing fibrosis associated proteins such as for example S100A4 (S100 calcium-binding protein A4) and ACTA2 (Actin, Alpha 2, Smooth Muscle, Aorta) in the target cell. In alternative embodiments, the polypeptide is capable or increasing expression of fibrosis associated proteins. In some embodiments, the target cell is a fibroblast, a scar derived fibroblast, a keloid scar derived fibroblast, a hypertrophic scar derived fibroblast, a fibrotic cell, a cancel cell, and a myofibroblast.

In some embodiments, the polypeptide is capable of modulating expression of extracellular matrix proteins involved in ECM organization, repair, renewal and/or remodeling. In other embodiments, the polypeptide is capable of modulating expression of a genetic signature comprising genes selected from FGFR1 (Fibroblast growth factor receptor 1), MMP1, MMP3, TIMP1, FGF7 (Fibroblast growth factor 7), TP63 (Tumor protein p63), SOD2 (Superoxide dismutase 2, mitochondrial), UBD (Ubiquitin D), HAS2, HAS3 (Hyaluronan synthase), ADAMTS1 (A disintegrin and metalloproteinase with thrombospondin motifs 1), IGF-1 (Insulin-like growth factor 1), IL-6, IL-32 (Interleukin), CCL2 (chemokine (C-C motif) ligand 2), BDKRB1 (Bradykinin receptor B1), S1000A4 and ACTA2.

In some embodiments, the polypeptide is capable of modulating genes selected from FGFR1 MMP1, MMP3, TIMP1, FGF7, and TP63 in a target cell. In some embodiments, the target cell comprises a fibrotic cell, a scar derived fibroblast.

In other embodiments, the polypeptide is capable of modulating procollagen peptides secretion from a target cell. In some embodiments, the polypeptide is capable of reducing expression and/or secretion of collagen and/or proteins involved in promoting contractility from a target cell. In some embodiments, the target cell comprises a normal fibroblast, a scar derived fibroblast, a fibrotic cell, a fibrotic cancer cell, or combinations thereof.

In another embodiment, the polypeptide is capable of reducing contractility of a target cell, such as for example a dermal scar fibroblast, thereby reducing scar formation of a target cell.

In another embodiment, the polypeptide is capable of modulating expression of fibrosis associated proteins from a target cell. In some embodiments, the fibrosis associated protein is selected from the group of fibronectin, collagen, lactotransferrin, thrombospondin-1, MMP1, MMP3 and TIMP1. In one embodiment, the target cell is selected from the group of a fibroblast, a scar derived fibroblast, a fibrotic cell and a cancer cell. In some embodiments, the polypeptide is capable of reducing production and/or secretion of fibronectin, collagen, thrombospondin-1 and lactotransferrin. In other embodiments, the polypeptide is capable of increasing production and/or secretion of MMP1, TIMP1.

In another embodiment, the polypeptide lacks botulinum toxin endopeptidase activity.

In still another embodiment, the polypeptide lacks botulinum toxin translocation activity. In one embodiment, the polypeptide lacks an amino acid sequence which is substantially identical to the amino acid sequence in the amino terminus of the heavy chain (H_(N)) of the botulinum toxin. In still another embodiment, the polypeptide consists of an amino acid sequence substantially identical to an amino acid sequence in a binding domain of a botulinum toxin.

In still another embodiment, the polypeptide comprises an amino acid sequence having at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% homology to a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 19, SEQ ID NOs: 3-5, SEQ ID NO: 6, SEQ ID NO: 20, SEQ ID Nos: 7-10, SEQ ID NO: 11, SEQ ID NO: 21, and SEQ ID NOs: 12-18.

In yet another embodiment, the polypeptide has at least about 90% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 19, SEQ ID NOs: 3-5, SEQ ID NO: 6, SEQ ID NO: 20, SEQ ID Nos: 7-10, SEQ ID NO: 11, SEQ ID NO: 21, and SEQ ID NOs: 12-18.

In yet another embodiment, the polypeptide consists essentially of the polypeptide sequence set forth in SEQ ID NO: 1, SEQ ID NO: 19, SEQ ID NOs: 3-5, SEQ ID NO: 6, SEQ ID NO: 20, SEQ ID Nos: 7-10, SEQ ID NO: 11, SEQ ID NO: 21, and SEQ ID NOs: 12-18.

In some embodiments, the polypeptide comprises, consists essentially of, or consists of the polypeptide sequence set forth in SEQ ID NO: 1, SEQ ID NO: 19, SEQ ID NOs: 3-5, SEQ ID NO: 6, SEQ ID NO: 20, SEQ ID Nos: 7-10, SEQ ID NO: 11, SEQ ID NO: 21, and SEQ ID NOs: 12-18, with the proviso that the polypeptide contains 1, 2, 3, 4, or 5 mutations in the polypeptide sequence. Preferably, the mutant polypeptide contains 1, 2, 3, 4, or 5 mutations in the polypeptide sequence of SEQ ID NO: 1, e.g., a mutant polypeptide comprising, consisting essentially of, or consisting of the polypeptide sequence set forth in SEQ ID NO: 25, SEQ ID NO: 26 or SEQ ID NO: 27.

In another aspect, a fusion protein comprising the polypeptide described herein is provided.

In another aspect, a pharmaceutical composition is provided that comprises a polypeptide or a fusion protein as described herein and a pharmaceutically acceptable carrier.

In one embodiment, the pharmaceutical composition is formulated for topical or transdermal administration.

In yet another aspect, a kit is described that comprises the polypeptide or a fusion protein as described herein. In some embodiments, the kit further comprises and instructions for administration. In some embodiments, the kit comprises one or more packages.

In yet another aspect, a method for alleviating and/or reducing the occurrence of a condition or disorder associated with extracellular matrix (ECM) dysregulation; ECM disease; connective or epithelial tissue disease; endothelial diseases; and fibrotic diseases; preferably fibrotic diseases; and particularly fibrotic disease of the ECM, connective or epithelial tissue, or endothelial tissue is provided.

In some embodiments, the method comprises administering to the subject a composition comprising a polypeptide having an amino acid sequence with at least about 90% sequence identity to an amino acid sequence in the full length of a botulinum toxin. In some embodiments, the method comprises administering to the subject a composition comprising a polypeptide having an amino acid sequence with at least about 90% sequence identity to an amino acid sequence in the binding domain of a botulinum toxin. In another embodiment, the method comprises administering to the subject a composition comprising a polypeptide having an amino acid sequence with at least about 90% sequence identity to an amino acid sequence in the amino or N-terminal half of the binding domain of the botulinum toxin (H_(CN)). In some embodiments, the polypeptide has a molecular weight between about 20 kDa to about 60 kDa. In one embodiment, modulating a skin quality attribute does not involve paralysis of a facial muscle. In some embodiments, the botulinum toxin is selected from the group consisting of Botulinum toxin serotype A (BoNT/A), Botulinum toxin serotype B (BoNT/B), Botulinum toxin serotype C₁ (BoNT/C₁), Botulinum toxin serotype D (BoNT/D), Botulinum toxin serotype E (BoNT/E), Botulinum toxin serotype F (BoNT/F), Botulinum toxin serotype G (BoNT/G), Botulinum toxin serotype H (BoNT/H), Botulinum toxin serotype X (BoNT/X), Enterococcus sp. Botulinum toxin J (eBoNT/J), and mosaic Botulinum toxins and/or variants thereof. Examples of mosaic toxins include BoNT/DC, BoNT/CD, and BoNT/FA. In one embodiment, the botulinum toxin is not Botulinum toxin serotype A (BoNT/A).

In still another aspect, a method for alleviating and/or reducing the occurrence of a fibrotic disorder in a subject in need thereof is provided. In some embodiments, the method comprises administering to the subject a composition comprising a polypeptide having an amino acid sequence with at least about 90% sequence identity to an amino acid sequence in the full length of a botulinum toxin. In some embodiments, the method comprises administering to the subject a composition comprising a polypeptide having an amino acid sequence with at least about 90% sequence identity to an amino acid sequence in the binding domain of a botulinum toxin. In another embodiment, the method comprises administering to the subject a composition comprising a polypeptide having an amino acid sequence with at least about 90% sequence identity to an amino acid sequence in the amino or N-terminal half of the binding domain of the botulinum toxin (H_(CN)). In some embodiments, the method comprises administering to the subject a composition comprising a polypeptide having an amino acid sequence with at least about 90% sequence identity to a binding domain of a botulinum toxin. In some embodiments, the method comprises administering to the subject a composition comprising a polypeptide having an amino acid sequence with at least about 90% sequence identity to the N-terminal half of the binding domain of a botulinum toxin (H_(CN)). In some embodiments, the polypeptide has a molecular weight between about 20 kDa to about 60 kDa. In one embodiment, treating or reducing the occurrence of the condition or disorder associated with ECM dysregulation does not involve paralysis of a target muscle.

In one embodiment, the fibrotic disorder is selected from the group consisting of idiopathic pulmonary fibrosis, kidney fibrosis, glomerulosclerosis, ocular fibrosis, osteoarthritis, scleroderma, cardiac fibrosis, and liver fibrosis. Fibrosis can occur in any organ or tissue including an organ selected from, but not limited to, kidney, eye, lung, liver, heart, and skin; or a tissue selected from, but not limited to connective, epithelial, and endothelial tissues. Connective tissues include, but are not limited to fibroblasts), osteoblasts, chondrocytes, myocytes and adipocytes.

In some embodiments, a method for treating, alleviating and/or reducing the occurrence of a scar in a subject in need thereof is provided. In some embodiments, the method comprises administering to the subject a composition comprising a polypeptide having an amino acid sequence with at least about 90% sequence identity to an amino acid sequence in the full length of a botulinum toxin. In some embodiments, the method comprises administering to the subject a composition comprising a polypeptide having an amino acid sequence with at least about 90% sequence identity to an amino acid sequence in the binding domain of a botulinum toxin. In another embodiment, the method comprises administering to the subject a composition comprising a polypeptide having an amino acid sequence with at least about 90% sequence identity to an amino acid sequence in the amino or N-terminal half of the binding domain of the botulinum toxin (H_(CN)). In some embodiments, the method comprises administering to the subject a composition comprising a polypeptide having an amino acid sequence with at least about 90% sequence identity to a binding domain of a botulinum toxin. In some embodiments, the method comprises administering to the subject a composition comprising a polypeptide having an amino acid sequence with at least about 90% sequence identity to the N-terminal half of the binding domain of a botulinum toxin (H_(CN)). In some embodiments, the polypeptide has a molecular weight between about 20 kDa to about 60 kDa. In one embodiment, treating or reducing the occurrence of the condition or disorder associated with ECM dysregulation does not involve paralysis of a target muscle.

In some embodiments, the scar is selected from the group selected from dermal scars, such as for example hypertrophic, keloid, atropic, stretch marks, capsular contracture, and umbilical scars, wound scars, surgical scars, such as for example scars resulted from after cesarean section, abdominoplasty, breast augmentation, abdominal surgery, face lift, or injection of fillers.

In some embodiments, a method for treating or reducing the occurrence of a fibrotic disorder associated with connective, epithelial, or endothelial, tissues in a subject in need thereof is provided. In some embodiments, the method comprises administering to the subject a composition comprising a polypeptide having an amino acid sequence with at least about 90% sequence identity to an amino acid sequence in the full length of a botulinum toxin. In some embodiments, the method comprises administering to the subject a composition comprising a polypeptide having an amino acid sequence with at least about 90% sequence identity to an amino acid sequence in the binding domain of a botulinum toxin. In another embodiment, the method comprises administering to the subject a composition comprising a polypeptide having an amino acid sequence with at least about 90% sequence identity to an amino acid sequence in the amino or N-terminal half of the binding domain of the botulinum toxin (H_(CN)). In some embodiments, the method comprises administering to the subject a composition comprising a polypeptide having an amino acid sequence with at least about 90% sequence identity to a binding domain of a botulinum toxin. In some embodiments, the method comprises administering to the subject a composition comprising a polypeptide having an amino acid sequence with at least about 90% sequence identity to the N-terminal half of the binding domain of a botulinum toxin (H_(CN)). In some embodiments, the polypeptide has a molecular weight between about 20 kDa to about 60 kDa. In one embodiment, treating or reducing the occurrence of the condition or disorder associated with ECM dysregulation does not involve paralysis of a target muscle.

In some embodiments, the disease or disorder associated with connective or epithelial or endothelial tissue fibroblasts include dermal scars (hypertrophic, keloid, atropic (for example with acne), stretch marks, and umbilical), wound scars, surgical scars (for example after cesarean section, abdominoplasty, breast augmentation, abdominal surgery, face lift, or injection of fillers), postoperative intraperitoneal adhesions, fibroma, liver fibrosis, cirrhosis, pancreatitis, benign prostatic hyperplasia, fibrotic bladder, interstitial cystitis, pulmonary fibrosis, kidney fibrosis, glomerulosclerosis, atrial fibrosis, endomyocardial fibrosis, glial scar, arterial stiffness, arthrofibrosis, cystic fibrosis, non-cystic fibrosis, Crohn's disease, Dupuytren's contracture, mediastinal fibrosis, myelofibrosis, Peyronie's disease, nephrogenic systemic fibrosis, progressive massive fibrosis, retroperitoneal fibrosis, adhesive capsulitis, mixed connective tissue disease, ocular fibrosis, osteoarthritis, scleroderma, asthma, and ocular fibrosis related conditions. Fibrosis can occur in any organ or tissue including an organ selected from, but not limited to, kidney, eye, liver, heart, and skin; or a tissue selected from, but not limited to connective, epithelial, or endothelial tissues

In other embodiments, the composition is formulated for topical, transdermal or intradermal administration.

In one embodiment, the polypeptide for use in any of the methods has at least about 90% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 1, 19, 3-18, and 25-27.

In other aspects, a polynucleotide is provided that is selected from the group consisting of:

-   -   (a) a cDNA which encodes a polypeptide comprising the binding         domain of a botulinum toxin, wherein the molecular weight of the         polypeptide is between about 1 kDa to about 90 kDa;     -   (b) a synthetic DNA which encodes a polypeptide comprising the         binding domain of a botulinum toxin, wherein the molecular         weight of the polypeptide is between about 1 kDa to about 90         kDa;     -   (c) a codon-optimized DNA which encodes a polypeptide comprising         the binding domain of a botulinum toxin, wherein the molecular         weight of the polypeptide is between about 1 kDa to about 90         kDa; and     -   (d) a DNA which is complementary to the DNA of any one of         (a)-(c).

In other aspects, a polynucleotide is provided that is selected from the group consisting of:

-   -   (a) a cDNA which encodes a polypeptide comprising the binding         domain of a botulinum toxin, wherein the molecular weight of the         polypeptide is less than 50 kDa;     -   (b) a synthetic DNA which encodes a polypeptide comprising the         binding domain of a botulinum toxin, wherein the molecular         weight of the polypeptide is less than 50 kDa;     -   (c) a codon-optimized DNA which encodes a polypeptide comprising         the binding domain of a botulinum toxin, wherein the molecular         weight of the polypeptide is less than 50 kDa; and     -   (d) a DNA which is complementary to the DNA of any one of         (a)-(c).

In other aspects, a polynucleotide is provided that is selected from the group consisting of:

-   -   (a) a cDNA which encodes a polypeptide comprising the binding         domain of a botulinum toxin, wherein the molecular weight of the         polypeptide is greater than 1 kDa but less than 15 kDa;     -   (b) a synthetic DNA which encodes a polypeptide comprising the         binding domain of a botulinum toxin, wherein the molecular         weight of the polypeptide is greater than 1 kDa but less than 15         kDa;     -   (c) a codon-optimized DNA which encodes a polypeptide comprising         the binding domain of a botulinum toxin, wherein the molecular         weight of the polypeptide is greater than 1 kDa but less than 15         kDa; and     -   (d) a DNA which is complementary to the DNA of any one of         (a)-(c).

In another still another aspect, a polynucleotide is provided which comprises at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or greater % sequence identity to the polynucleotide sequence of SEQ ID NO: 2 or a nucleic acid encoding a polypeptide selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 19, SEQ ID NOs: 3-5, SEQ ID NO: 6, SEQ ID NO: 20, SEQ ID Nos: 7-10, SEQ ID NO: 11, SEQ ID NO: 21, SEQ ID NOs: 12-18, and SEQ ID NOs: 25-27 or a degenerate thereof or an RNA equivalent thereof.

In yet another embodiment, the polynucleotide consists essentially of the nucleic acid sequence set forth in SEQ ID NO: 2 or a degenerate thereof or an RNA equivalent thereof.

In another aspect, a vector comprising a polynucleotide as described herein is provided. In other aspects, a host cell comprising the vector is provided. In one embodiment, the host cell is not Clostridium botulinum.

In other aspects, kits, compositions, and methods of using the kits and compositions are provided. In one embodiment, a kit is provided, comprising, in one or more packages, a vector and instructions for expressing the polynucleotide in a suitable host cell. In another embodiment, a composition comprising a vector or a host cell and a pharmaceutical excipient is provided. In still another embodiment, a method to treat and/or reduce the occurrence of a fibrotic disorder that comprises administering to a subject the composition comprising a vector or a host cell to achieve expression of the polypeptide is provided. In still another embodiment, a method for treating and/or reducing the occurrence of a scar that comprises administering to a subject the composition comprising a vector or a host cell to achieve expression of the polypeptide is provided.

BRIEF DESCRIPTION OF THE SEQUENCES SEQ ID NO: 1 is the amino acid sequence of the cell binding domain of BoNT/A1 (H_(C)/A): TSILNLRYESNHLIDLSRYASKINIGSKVNFDPIDKNQIQLFNLESSKIEV ILKNAIVYNSMYENFSTSFWIRIPKYFNSISLNNEYTIINCMENNSGWKVS LNYGEIIWTLQDTQEIKQRVVFKYSQMINISDYINRWIFVTITNNRLNNSK IYINGRLIDQKPISNLGNIHASNNIMFKLDGCRDTHRYIWIKYFNLFDKEL NEKEIKDLYDNQSNSGILKDFWGDYLQYDKPYYMLNLYDPNKYVDVNNVGI RGYMYLKGPRGSVMTTNIYLNSSLYRGTKFIIKKYASGNKDNIVRNNDRVY INVVVKNKEYRLATNASQAGVEKILSALEIPDVGNLSQVVVMKSKNDQGIT NKCKMNLQDNNGNDIGFIGFHQFNNIAKLVASNWYNRQIERSSRTLGCSWE FIPVDDGWGERPL SEQ ID NO: 19 is the amino acid sequence of the cell binding domain of BoNT/A1 (H_(C)/A) further comprising a N-terminal his-tag (amino acids which make up the N-terminal tag are underlined): MGSSHHHHHHSSGLVPRGSHMD TSILNLRYESNHLIDLSRYASKINIGSKV NFDPIDKNQIQLFNLESSKIEVILKNAIVYNSMYENFSTSFWIRIPKYFNS ISLNNEYTIINCMENNSGWKVSLNYGEIIWTLQDTQEIKQRVVFKYSQMIN ISDYINRWIFVTITNNRLNNSKIYINGRLIDQKPISNLGNIHASNNIMFKL DGCRDTHRYIWIKYFNLFDKELNEKEIKDLYDNQSNSGILKDFWGDYLQYD KPYYMLNLYDPNKYVDVNNVGIRGYMYLKGPRGSVMTTNIYLNSSLYRGTK FIIKKYASGNKDNIVRNNDRVYINVVVKNKEYRLATNASQAGVEKILSALE IPDVGNLSQVVVMKSKNDQGITNKCKMNLQDNNGNDIGFIGFHQFNNIAKL VASNWYNRQIERSSRTLGCSWEFIPVDDGWGERPLQ SEQ ID NO: 2 is the DNA sequence of the cell binding domain of BoNT/A1: accagcattctgaacctgcgttatgaaagcaaccatctgattgatctgagc cgttatgcgagcaaaattaacattggcagcaaagtgaactttgatccgatt gataagaaccagattcagctgtttaacctggaaagcagcaaaattgaagtg attctgaagaacgcgattgtgtataacagcatgtatgaaaactttagcacc agcttttggattcgtattccgaaatattttaacagcattagcctgaacaac gaatataccattattaactgcatggaaaacaacagcggctggaaagtgagc ctgaactatggcgaaattatttggaccctgcaggatacccaggaaattaaa cagcgtgtggtgtttaaatatagccagatgattaacattagcgattatatt aaccgttggatctttgtgaccattaccaacaaccgtctgaacaacagcaaa atttatattaacggccgtctgattgatcagaaaccgattagcaacctgggc aacattcatgcgagcaacaacattatgtttaaactggatggctgccgtgat acccatcgttatatttggattaaatattttaacctgtttgataaagagctc aacgagaaagaaattaaagatctgtatgataaccagagcaacagcggcatt ctgaaagatttctggggcgattatctgcagtatgataaaccgtattatatg ctgaacctgtatgatccgaacaaatatgtggatgtgaacaacgtgggcatt cgtggctatatgtatctgaaaggcccgcgtggcagcgtgatgaccaccaac atttatctgaacagcagcctgtatcgtggcaccaaatttattattaagaag tatgcgagcggcaacaaagataacattgtgcgtaacaacgatcgtgtgtat attaacgtggtggtgaagaacaaagaatatcgtctggcgaccaacgcgagc caggcgggcgtggaaaagattctgagcgcgctggaaattccggatgtgggc aacctgagccaggtggtggtgatgaaaagcaagaacgatcagggcattacc aacaaatgcaaaatgaacctgcaggataacaacggcaacgatattggcttt attggctttcatcagtttaacaacattgcgaaactggtggcgagcaactgg tataaccgtcagattgaacgtagcagccgtaccctgggctgcagctgggaa tttattccggtggatgatggctggggcgaacgtccgctgtaa SEQ ID NO: 3 is an amino acid sequence of the binding domain region within the heavy chain of BoNT/B, H_(C)/B, (GENBANK Accession No. BAE48264): NIILNLRYKDNNLIDLSGYGAKVEVYDGVELNDKNQFKLTSSANSKIRVTQ NQNIIFNSVFLDFSVSFWIRIPKYKNDGIQNYIHNEYTIINCMKNNSGWKI SIRGNRIIWTLIDINGKTKSVFFEYNIREDISEYINRWFFVTITNNLNNAK IYINGKLESNTDIKDIREVIANGEIIFKLDGDIDRTQFIWMKYFSIFNTEL SQSNIEERYKIQSYSEYLKDFWGNPLMYNKEYYMFNAGNKNSYIKLKKDSP VGEILTRSKYNQNSKYINYRDLYIGEKFIIRRKSNSQSINDDIVRKEDYIY LDFFNLNQEWRVYTYKYFKKEEEKLFLAPISDSDEFYNTIQIKEYDEQPTY SCQLLFKKDEESTDEIGLIGIHRFYESGIVFEEYKDYFCISKWYLKEVKRK PYNLKLGCNWQFIPKDEGWTE SEQ ID NO: 4 is an amino acid sequence of the binding domain region within the heavy chain of BoNT/C, H_(C)/C, (GENBANK Accession No. P18640): SKILSLQNRKNTLVDTSGYNAEVSEEGDVQLNPIFPFDFKLGSSGEDRGKV IVTQNENIVYNSMYESFSISFWIRINKWVSNLPGYTIIDSVKNNSGWSIGI ISNFLVFTLKQNEDSEQSINFSYDISNNAPGYNKWFFVTVTNNMMGNMKIY INGKLIDTIKVKELTGINFSKTITFEINKIPDTGLITSDSDNINMWIRDFY IFAKELDGKDINILFNSLQYTNVVKDYWGNDLRYNKEYYMVNIDYLNRYMY ANSRQIVFNTRRNNNDFNEGYKIIIKRIRGNTNDTRVRGGDILYFDMTINN KAYNLFMKNETMYADNHSTEDIYAIGLREQTKDINDNIIFQIQPMNNTYYY ASQIFKSNFNGENISGICSIGTYRFRLGGDWYRHNYLVPTVKQGNYASLLE STSTHWGFVPVSE SEQ ID NO: 5 is an amino acid sequence of the binding domain region within the heavy chain of BoNT/D, H_(C)/D, (GENBANK Accession No. P19321): SKILSLQNKKNALVDTSGYNAEVRVGDNVQLNTIYTNDFKLSSSGDKIIVN LNNNILYSAIYENSSVSFWIKISKDLTNSHNEYTIINSIEQNSGWKLCIRN GNIEWILQDVNRKYKSLIFDYSESLSHTGYTNKWFFVTITNNIMGYMKLYI NGELKQSQKIEDLDEVKLDKTIVFGIDENIDENQMLWIRDFNIFSKELSNE DINIVYEGQILRNVIKDYWGNPLKFDTEYYTINDNYIDRYIAPESNVLVLV QYPDRSKLYTGNPITIKSVSDKNPYSRILNGDNIILHMLYNSRKYMIIRDT DTIYATQGGECSQNCVYALKLQSNLGNYGIGIFSIKNIVSKNKYCSQIFSS FRENTMLLADIYKPWRFSFKNAYTPVAVTNYETKLLSTSSFWKFISRDPGW VE SEQ ID NO: 6 is an amino acid sequence of the binding domain region within the heavy chain of BoNT/DC, H_(C)/DC, (GENBANK Accession No. EF378947): SKILSLQNKKNTLMDTSGYNAEVRVEGNVQLNPIFPFDFKLGSSGDDRGKV IVTQNENIVYNAMYESFSISFWIRINKWVSNLPGYTIIDSVKNNSGWSIGI ISNFLVFTLKQNENSEQDINFSYDISKNAAGYNKWFFVTITTNMMGNMMIY INGKLIDTIKVKELTGINFSKTITFQMNKIPNTGLITSDSDNINMWIRDFY IFAKELDDKDINILFNSLQYTNVVKDYWGNDLRYDKEYYMINVNYMNRYMS KKGNGIVFNTRKNNNDFNEGYKIIIKRIRGNTNDTRVRGENVLYFNTTIDN KQYSLGMYKPSRNLGTDLVPLGALDQPMDEIRKYGSFIIQPCNTFDYYASQ LFLSSNATTNRLGILSIGSYSFKLGDDYWFNHEYLIPVIKIEHYASLLEST STHWVFVPASE SEQ ID NO: 20 is an amino acid sequence of the binding domain region within the heavy chain of BoNT/DC further comprising a N-terminal his-tag (amino acids which make up the N-terminal tag are underlined) (GENBANK Accession No. EF378947): MGSSHHHHHHSSGLVPRGSHMD SKILSLQNKKNTLMDTSGYNAEVRVEGNV QLNPIFPFDFKLGSSGDDRGKVIVTQNENIVYNAMYESFSISFWIRINKWV SNLPGYTIIDSVKNNSGWSIGIISNFLVFTLKQNENSEQDINFSYDISKNA AGYNKWFFVTITTNMMGNMMIYINGKLIDTIKVKELTGINFSKTITFQMNK IPNTGLITSDSDNINMWIRDFYIFAKELDDKDINILFNSLQYTNVVKDYWG NDLRYDKEYYMINVNYMNRYMSKKGNGIVFNTRKNNNDFNEGYKIIIKRIR GNTNDTRVRGENVLYFNTTIDNKQYSLGMYKPSRNLGTDLVPLGALDQPMD EIRKYGSFIIQPCNTFDYYASQLFLSSNATTNRLGILSIGSYSFKLGDDYW FNHEYLIPVIKIEHYASLLESTSTHWVFVPASEQ SEQ ID NO: 7 is an amino acid sequence of the binding domain region within the heavy chain of BoNT/E, H_(C)/E, (GENBANK Accession No. AFV91344): SSVLNMRYKNDKYVDTSGYDSNININGDVYKYPTNKNQFGIYNDKLSEVNI SQNDYIIYDNKYKNFSISFWVRIPNYDNKIVNVNNEYTIINCMRDNNSGWK VSLNHNEIIWTLQDNAGINQKLAFNYGNANGISDYINKWIFVTITNDRLGD SKLYINGNLIDQKSILNLGNIHVSDNILFKIVNCSYTRYIGIRYFNVFDKE LDETEIQTLYSNEPNTNILKDFWGNYLLYDKEYYLLNVLKPNNFIDRRKDS TLSINNIRSTILLANRLYSGIKVKIQRVNNSSTNDNLVRKNDQVYINFVAS KTHLFPLYADTATTNKEKTIKISSSGNRFNQVVVMNSVGNNCTMNFKNNNG NNIGLLGFKADTVVASTWYYTHMRDHTNSNGCFWNFISEEHGWQEK SEQ ID NO: 8 is an amino acid sequence of the binding domain region within the heavy chain of BoNT/F, H_(C)/F, (GENBANK Accession No. ABS41202): NSILDMRYENNKFIDISGYGSNISINGDVYIYSTNRNQFGIYSSKPSEVNI AQNNDIIYNGRYQNFSISFWVRIPKYFNKVNLNNEYTIIDCIRNNNSGWKI SLNYNKIIWTLQDTAGNNQKLVFNYTQMISISDYINKWIFVTITNNRLGNS RIYINGNLIDEKSISNLGDIHVSDNILFKIVGCNDTRYVGIRYFKVFDTEL GKTEIETLYSDEPDPSILKDFWGNYLLYNKRYYLLNLLRTDKSITQNSNFL NINQQRGVYQKPNIFSNTRLYTGVEVIIRKNGSTDISNTDNFVRKNDLAYI NVVDRDVEYRLYADISIAKPEKIIKLIRTSNSNNSLGQIIVMDSIGNNCTM NFQNNNGGNIGLLGFHSNNLVASSWYYNNIRKNTSSNGCFWSFISKEHGWQ EN SEQ ID NO: 9 is an amino acid sequence of the binding domain region within the heavy chain of BoNT/G, H_(C)/G, (GENBANK Accession No. X74162): NAILSLSYRGGRLIDSSGYGATMNVGSDVIFNDIGNGQFKLNNSENSNITA HQSKFVVYDSMFDNFSINFWVRTPKYNNNDIQTYLQNEYTIISCIKNDSGW KVSIKGNRIIWTLIDVNAKSKSIFFEYSIKDNISDYINKWFSITITNDRLG NANIYINGSLKKSEKILNLDRINSSNDIDFKLINCTDTTKFVWIKDFNIFG RELNATEVSSLYWIQSSTNTLKDFWGNPLRYDTQYYLFNQGMQNIYIKYFS KASMGETAPRTNFNNAAINYQNLYLGLRFIIKKASNSRNINNDNIVREGDY IYLNIDNISDESYRVYVLVNSKEIQTQLFLAPINDDPTFYDVLQIKKYYEK TTYNCQILCEKDTKTFGLFGIGKFVKDYGYVWDTYDNYFCISQWYLRRISE NINKLRLGCNWQFIPVDEGWTE SEQ ID NO: 10 is an amino acid sequence of the binding domain region within the heavy chain of BoNT/X, H_(C)/X, (GENBANK Accession No. BAQ12790): VLNLGAEDGKIKDLSGTTSDINIGSDIELADGRENKAIKIKGSENSTIKIA MNKYLRFSATDNFSISFWIKHPKPTNLLNNGIEYTLVENFNQRGWKISIQD SKLIWYLRDHNNSIKIVTPDYIAFNGWNLITITNNRSKGSIVYVNGSKIEE KDISSIWNTEVDDPIIFRLKNNRDTQAFTLLDQFSIYRKELNQNEVVKLYN YYFNSNYIRDIWGNPLQYNKKYYLQTQDKPGKGLIREYWSSFGYDYVILSD SKTITFPNNIRYGALYNGSKVLIKNSKKLDGLVRNKDFIQLEIDGYNMGIS ADRFNEDTNYIGTTYGTTHDLTTDFEIIQRQEKYRNYCQLKTPYNIFHKSG LMSTETSKPTFHDYRDWVYSSAWYFQNYENLNLRKHTKTNWYFIPKDEGWD ED SEQ ID NO: 11 is an amino acid sequence corresponding to residues 1-218 of the amino acid sequence of the cell binding domain of BoNT/A1, referred to as the N-terminal fragment of the cell binding domain and abbreviated HCN/A (PDB ID: 3BTA, 4JRA): TSILNLRYESNHLIDLSRYASKINIGSKVNFDPIDKNQIQLFNLESSKIEV ILKNAIVYNSMYENFSTSFWIRIPKYFNSISLNNEYTIINCMENNSGWKVS LNYGEIIWTLQDTQEIKQRVVFKYSQMINISDYINRWIFVTITNNRLNNSK IYINGRLIDQKPISNLGNIHASNNIMFKLDGCRDTHRYIWIKYFNLFDKEL NEKEIKDLYDNQSN SEQ ID NO: 21 is an amino acid sequence that includes an N-terminal histidine tag and residues 1-218 of the amino acid sequence of the cell binding domain of BoNT/A1 (H_(C)/A) (amino acids which make up the N-terminal tag are underlined): MGSSHHHHHHSSGLVPRGSHMD TSILNLRYESNHLIDLSRYASKINIGSKV NFDPIDKNQIQLFNLESSKIEVILKNAIVYNSMYENFSTSFWIRIPKYFNS ISLNNEYTIINCMENNSGWKVSLNYGEIIWTLQDTQEIKQRVVFKYSQMIN ISDYINRWIFVTITNNRLNNSKIYINGRLIDQKPISNLGNIHASNNIMFKL DGCRDTHRYIWIKYFNLFDKELNEKEIKDLYDNQSN SEQ ID NO: 12 is an amino acid sequence from the N- terminal region of the binding domain of BoNT/B, H_(CN)/B (PDB ID: 2NM1): NIILNLRYKDNNLIDLSGYGAKVEVYDGVELNDKNQFKLTSSANSKIRVTQ NQNIIFNSVFLDFSVSFWIRIPKYKNDGIQNYIHNEYTIINCMKNNSGWKI SIRGNRIIWTLIDINGKTKSVFFEYNIREDISEYINRWFFVTITNNLNNAK IYINGKLESNTDIKDIREVIANGETIFKLDGDIDRTQFIWMKYFSIFNTEL SQSNIEERYKIQSYSEY SEQ ID NO: 13 is an amino acid sequence from the N- terminal region of the binding domain of BoNT/C, H_(CN)/C (PDB ID: 3R4U) SKILSLQNRKNTLVDTSGYNAEVSEEGDVQLNPIFPFDFKLGSSGEDRGKV IVTQNENIVYNSMYESFSISFWIRINKWVSNLPGYTIIDSVKNNSGWSIGI ISNFLVFTLKQNEDSEQSINFSYDISNNAPGYNKWFFVTVTNNMMGNMKIY INGKLIDTIKVKELTGINFSKTITFEINKIPDTGLITSDSDNINMWIRDFY IFAKELDGKDINILFNSLQYTN SEQ ID NO: 14 is an amino acid sequence from the N- terminal region of the binding domain of BoNT/D, H_(CN)/D (PDB ID: 3N7J): SKILSLQNKKNALVDTSGYNAEVRVGDNVQLNTIYTNDFKLSSSGDKIIVN LNNNILYSAIYENSSVSFWIKISKDLTNSHNEYTIINSIEQNSGWKLCIRN GNIEWILQDVNRKYKSLIFDYSESLSHTGYTNKWFFVTITNNIMGYMKLYI NGELKQSQKIEDLDEVKLDKTIVFGIDENIDENQMLWIRDFNIFSKELSNE DINIVYEGQIL SEQ ID NO: 15 is an amino acid sequence from the N- terminal region of the binding domain of BoNT/DC H_(CN)/DC (PDB ID: 4ISQ): SKILSLQNKKNTLMDTSGYNAEVRVEGNVQLNPIFPFDFKLGSSGDDRGKV IVTQNENIVYNAMYESFSISFWIRINKWVSNLPGYTIIDSVKNNSGWSIGI ISNFLVFTLKQNENSEQDINFSYDISKNAAGYNKWFFVTITTNMMGNMMIY INGKLIDTIKVKELTGINFSKTITFQMNKIPNTGLITSDSDNINMWIRDFY IFAKELDDKDINILFNSLQYTN SEQ ID NO: 16 is an amino acid sequence from the N- terminal region of the binding domain of BoNT/E, H_(CN)/E (PDB: 4ZKT): SSVLNIVIRYKNDKYVDTSGYDSNININGDVYKYPTNKNQFGIYNDKLSEV NISQNDYIIYDNKYKNFSISFWVRIPNYDNKIVNVNNEYTIINCMRDNNSG WKVSLNHNEIIWTLQDNAGINQKLAFNYGNANGISDYINKWIFVTITNDRL GDSKLYINGNLIDQKSILNLGNIHVSDNILFKIVNCSYTRYIGIRYFNVFD KELDETEIQTLYSNEPNTN SEQ ID NO: 17 is an amino acid sequence from the N- terminal region of the binding domain of BoNT/F, H_(CN)/F (PDB ID: 3RSJ): NSILDMRYENNKFIDISGYGSNISINGDVYIYSTNRNQFGIYSSKPSEVNI AQNNDIIYNGRYQNFSISFWVRIPKYFNKVNLNNEYTIIDCIRNNNSGWKI SLNYNKIIWTLQDTAGNNQKLVFNYTQMISISDYINKWIFVTITNNRLGNS RIYINGNLIDEKSISNLGDIHVSDNILFKIVGCNDTRYVGIRYFKVFDTEL GKTEIETLYSDEPD SEQ ID NO: 18 is an amino acid sequence from the N- terminal region of the binding domain of BoNT/G, H_(CN)/G (PDB ID: 2VXR) NAILSLSYRGGRLIDSSGYGATMNVGSDVIFNDIGNGQFKLNNSENSNITA HQSKFVVYDSMFDNFSINFWVRTPKYNNNDIQTYLQNEYTIISCIKNDSGW KVSIKGNRIIWTLIDVNAKSKSIFFEYSIKDNISDYINKWFSITITNDRLG NANIYINGSLKKSEKILNLDRINSSNDIDFKLINCTDTTKFVWIKDFNIFG RELNATEVSSLYWIQSSTNT

BRIEF DESCRIPTION OF THE DRAWINGS

The details of one or more embodiments of the disclosure are set forth in the accompanying drawings/tables and the description below. Other features, objects, and advantages of the disclosure will be apparent from the drawings/tables and detailed description, and from the claims.

FIG. 1 is a bar graph showing the fold-change in expression of the indicated genes in normal human primary fibroblast cells after treatment with 10 nM (solid bar), 100 nM (unfilled bar), or 1 μM (hatched bar) of a polypeptide having an amino acid sequence of SEQ ID NO: 19 for 1 day (24 hours), where the fold-change is expressed relative to untreated control cells;

FIG. 2 is a bar graph showing the fold-change in expression of the indicated genes in keloid scar human primary fibroblast cells after treatment with 1 nM (horizontal line fill), 10 nM (unfilled bar), 100 nM (hatched bar) or 1 μM (solid bar) of a polypeptide having an amino acid sequence of SEQ ID NO: 21 for 24 hours, where the fold-change is expressed relative to untreated control cells.

FIG. 3 is a bar graph showing the amount (ng/mL) of Procollagen Type I C-Peptide (PIP) secreted into the media from keloid scar human primary fibroblast cells after treatment with 1 μM of a polypeptide having an amino acid sequence of SEQ ID NO: 19.

FIG. 4 is a bar graph showing the size (cm²) of a fibrin hydrogel with keloid scar human primary fibroblast cells, which is a measure for fibroblast contractility and fibroblast-myofibroblast transition, after treatment with 1 μM of a polypeptide having an amino acid sequence of SEQ ID NO: 19.

FIGS. 5A-5E are bar graphs showing the amount (ng/mL) of fibronectin, collagen IV, thrombospondin-1, MMP1 and TIMP1, respectively, secreted into the media from fibrotic MDA-231 cells, where fibrosis was induced by hypoxia, after treatment with 20 pM, 0.1 μM or 1 μM of a polypeptide having an amino acid sequence of SEQ ID NO: 19 or a polypeptide having an amino acid sequence of SEQ ID NO: 21.

FIG. 6. is a bar graph showing the normalized percentage of fibronectin secreted into the media from keloid scar fibroblast (KF116R) cells after treatment with 0 (media only control, unfilled bar), 20 pM (horizontal line fill), 200 pM (vertical line fill), 20 nM (hatched bar), or 200 nM (solid bar) of a polypeptide having an amino acid sequence of SEQ ID NO: 19 for 1, 4, 7 or 9 days. The asterisk indicates statistical significance of p<0.05 compared to media only control.

FIG. 7 is a bar graph showing the amount of protein detected by Mass Spectroscopy (MS) protein analysis in the media collected from normal human fibroblasts (HDFa) after treatment with 20 pM of polypeptide having an amino acid sequence of SEQ ID NO: 19 (solid bar).

FIG. 8 is a bar graph showing the fold-change in expression of the indicated genes in keloid scar human primary fibroblast cells after treatment with 0.1 μM (solid bar) or 1 μM (hatched bar) of a polypeptide having an amino acid sequence of SEQ ID NO: 20; or 1 μM (unfilled bar) of a polypeptide having an amino acid sequence of SEQ ID NO: 21.

DETAILED DESCRIPTION

Various aspects will be described more fully hereinafter. Such aspects may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will be thorough and complete.

Definitions

Where a range of values is provided in this disclosure, it is intended that each intervening value between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the disclosure. For example, if a range of 1 μM to 8 μM is stated, it is intended that 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, and 7 μM are also explicitly disclosed, as well as the range of values greater than or equal to 1 μM and the range of values less than or equal to 8 μM.

The singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to an “amino acid” includes a single amino acid as well as two or more of the same or different amino acids.

The word “about” means a range of plus or minus 10% of that value, e.g., “about 50” means 45 to 55, “about 25,000” means 22,500 to 27,500, etc., unless the context of the disclosure indicates otherwise, or is inconsistent with such an interpretation. For example, in a list of numerical values such as “about 49, about 50, about 55, “about 50” means a range extending to less than half the interval(s) between the preceding and subsequent values, e.g., more than 49.5 to less than 52.5.

“Administration”, or “to administer” means the step of giving (i.e. administering) a pharmaceutical composition to a subject, or alternatively a subject receiving a pharmaceutical composition. The pharmaceutical compositions disclosed herein can be locally administered by various methods. For example, intramuscular, intradermal, subcutaneous administration, intrathecal administration, intraperitoneal administration, topical (transdermal), instillation, and implantation (for example, of a slow-release device such as polymeric implant or miniosmotic pump) can all be appropriate routes of administration.

“Alleviating” means a reduction in the occurrence of a pain, of a headache, or of any symptom or cause of a condition or disorder. Thus, alleviating includes some reduction, significant reduction, near total reduction, and total reduction.

The term “amino acid” means a naturally occurring or synthetic amino acid, as well as amino acid analogs, stereoisomers, and amino acid mimetics that function similarly to the naturally occurring amino acids. Included by this definition are natural amino acids such as: (1) histidine (His; H) (2) isoleucine (Ile; I) (3) leucine (Leu; L) (4) Lysine (Lys; K) (5) methionine (Met; M) (6) phenylalanine (Phe; F) (7) threonine (Thr; T) (8) tryptophan (Trp; W) (9) valine (Val; V) (10) arginine (Arg; R) (11) cysteine (Cys; C) (12) glutamine (Gln; Q) (13) glycine (Gly; G) (14) proline (Pro; P) (15) serine (Ser; S) (16) tyrosine (Tyr; Y) (17) alanine (Ala; A) (18) asparagine (Asn; N) (19) aspartic acid (Asp; D) (20) glutamic acid (Glu; E) (21) selenocysteine (Sec; U); including unnatural amino acids: (a) citrulline (Cit); (b) cystine; (c) gama-amino butyric acid (GABA); (d) ornithine (Orn); (f) theanine; (g) homocysteine (Hey); (h) thyroxine (Thx); and amino acid derivatives such as betaine; carnitine; carnosine creatine; hydroxytryptophan; hydroxyproline (Hyp); N-acetyl cysteine; S-Adenosyl methionine (SAM-e); taurine; tyramine.

“Amino acid residue” means the individual amino acid units incorporated into a polypeptide.

“Animal protein free” means the absence of blood derived, blood pooled and other animal derived products or compounds. “Animal” means a mammal (such as a human), bird, reptile, fish, insect, spider or other animal species. “Animal” excludes microorganisms, such as bacteria. Thus, an animal protein free pharmaceutical composition can include a botulinum neurotoxin. For example, an “animal protein free” pharmaceutical composition means a pharmaceutical composition which is either substantially free or essentially free or entirely free of a serum derived albumin, gelatin and other animal derived proteins, such as immunoglobulins. An example of an animal protein free pharmaceutical composition is a pharmaceutical composition which comprises or which consists of a botulinum toxin (as the active ingredient) and a suitable polysaccharide as a stabilizer or excipient.

“Binding domain” of a toxin as used herein encompasses the wild type binding domain, variants and/or fragments thereof.

“Botulinum toxin” means a neurotoxin produced by Clostridium botulinum, as well as a botulinum toxin (or the light chain or the heavy chain thereof) made recombinantly by a non-Clostridial species. The phrase “botulinum toxin”, as used herein, encompasses the botulinum toxin serotypes A, B, C, D, E, F, G, H and X, and their subtypes, mosaic toxins, such as BoNT/DC and BoNT/CD, and any other types of subtypes thereof, or any re-engineered proteins, analogs, derivatives, homologs, parts, sub-parts, variants, or versions, in each case, of any of the foregoing. “Botulinum toxin”, as used herein, also encompasses a “modified botulinum toxin”. Further “botulinum toxin” as used herein also encompasses a botulinum toxin complex, (for example, the 300, 600 and 900 kDa complexes), as well as the neurotoxic component of the botulinum toxin (150 kDa) that is unassociated with the complex proteins.

“Clostridial toxin” refers to any toxin produced by a Clostridial toxin strain that can execute the overall cellular mechanism whereby a Clostridial toxin intoxicates a cell and encompasses the binding of a Clostridial toxin to a low or high affinity Clostridial toxin receptor, the internalization of the toxin/receptor complex, the translocation of the Clostridial toxin light chain into the cytoplasm and the enzymatic modification of a Clostridial toxin substrate. Non-limiting examples of Clostridial toxins include Botulinum toxins, such as a BoNT/A, a BoNT/B, a BoNT/C₁, a BoNT/D, a BoNT/CD, a BoNT/DC, a BoNT/E, a BoNT/F, a BoNT/G, a BoNT/H (aka type FA or HA), a BoNT/X, eBoNT/J, a Tetanus toxin (TeNT), a Baratii toxin (BaNT), and a Butyricum toxin (BuNT). The BoNT/C2 cytotoxin and BoNT/C3 cytotoxin, not being neurotoxins, are excluded from the term “Clostridial toxin.” The term Clostridial toxin also includes the approximately 150-kDa Clostridial toxin alone (i.e. without the NAPs). A Clostridial toxin includes naturally occurring Clostridial toxin variants, such as, e.g., Clostridial toxin isoforms and Clostridial toxin subtypes; non-naturally occurring Clostridial toxin variants, such as, e.g., conservative Clostridial toxin variants, non-conservative Clostridial toxin variants, Clostridial toxin chimeric variants and active Clostridial toxin fragments thereof, or any combination thereof. A Clostridial toxin also includes Clostridial toxin complexes, which refers to a complex comprising a Clostridial toxin and non-toxin associated proteins (NAPs), such as, e.g., a Botulinum toxin complex, a Tetanus toxin complex, a Baratii toxin complex, and a Butyricum toxin complex. Non-limiting examples of Clostridial toxin complexes include those produced by a Clostridium botulinum, such as, e.g., a 900-kDa BoNT/A complex, a 500-kDa BoNT/A complex, a 300-kDa BoNT/A complex, a 500-kDa BoNT/B complex, a 500-kDa BoNT/C₁ complex, a 500-kDa BoNT/D complex, a 300-kDa BoNT/D complex, a 300-kDa BoNT/E complex, and a 300-kDa BoNT/F complex.

“Cellular phenotype” refers to any change in gene expression, protein expression, synthesis of factors, and/or secretion of proteins and factors that affect the structure and/or function of a cell and/or the tissue that the cell is a part of, including extracellular matrix structure and sebum.

“Effective amount” as applied to the biologically active ingredient means that amount of the ingredient which is generally sufficient to affect a desired change in the subject. For example, where the desired effect is a reduction in expression of fibrosis associated proteins, an effective amount of the ingredient is that amount which causes at least a substantial reduction of in expression of fibrosis associated proteins, and without resulting in significant toxicity. In other aspects of this embodiment, a therapeutically effective concentration of a Clostridial toxin active ingredient reduces a symptom associated with the aliment being treated by, e.g., at most 10%, at most 20%, at most 30%, at most 40%, at most 50%, at most 60%, at most 70%, at most 80%, at most 90% or at most 100%

“Heavy chain” means the heavy chain of a botulinum neurotoxin. It has a molecular weight of about 100 kDa and can be referred to as the H chain, or as H.

H_(C) means a fragment (about 50 kDa) derived from the H chain of a botulinum neurotoxin which is approximately equivalent to the carboxyl end segment of the H chain, or the portion corresponding to that fragment in the intact H chain. It is believed to contain the portion of the natural or wild type botulinum neurotoxin involved in high affinity, presynaptic binding to motor neurons.

H_(N) means a fragment (about 50 kDa) derived from the H chain of a botulinum neurotoxin which is approximately equivalent to the amino end segment of the H chain, or the portion corresponding to that fragment in the intact in the H chain. It is believed to contain the portion of the natural or wild type botulinum neurotoxin involved in the translocation of the L chain across an intracellular endosomal membrane.

“Homolog” means a protein in a group of proteins that perform the same biological function, e.g., proteins that belong to the same Clostridial toxin family and that provide a common activity, e.g., receptor-binding activity. Homologs are generally expressed by homologous genes.

“Isolated” means a nucleic acid sequence or a polypeptide sequence that is separated from the wild or native sequence in which it naturally occurs or is in an environment different from that in which the sequence naturally occurs.

“Light chain” means the light chain of a Clostridial neurotoxin. It has a molecular weight of about 50 kDa, and can be referred to as the L chain, L, or as the proteolytic domain (amino acid sequence) of a botulinum neurotoxin.

LH_(N) or L-H_(N) means a fragment derived from a Clostridial neurotoxin that contains the L chain, or a functional fragment thereof coupled to the H_(N) domain. It can be obtained from the intact Clostridial neurotoxin by proteolysis, so as to remove or to modify the H_(C) domain.

“Implant” means a controlled release (e.g., pulsatile or continuous) composition or drug delivery system. The implant can be, for example, injected, inserted or implanted into a human body.

“Local administration” means direct administration of a pharmaceutical at or to the vicinity of a site on or within an animal body, at which site a biological effect of the pharmaceutical is desired, such as via, for example, intramuscular or intra- or subdermal injection or topical administration. Local administration excludes systemic routes of administration, such as intravenous or oral administration. Topical administration is a type of local administration in which a pharmaceutical agent is applied to a patient's skin.

“Modified botulinum toxin” means a botulinum toxin that has had at least one of its amino acids deleted, modified, or replaced, as compared to a native botulinum toxin. Additionally, the modified botulinum toxin can be a recombinantly produced neurotoxin, or a derivative or fragment of a recombinantly made neurotoxin. A modified botulinum toxin retains at least one biological activity of the native botulinum toxin, such as, the ability to bind to a botulinum toxin receptor, or the ability to inhibit neurotransmitter release from a neuron. One example of a modified botulinum toxin is a botulinum toxin that has a light chain from one botulinum toxin serotype (such as serotype A), and a heavy chain from a different botulinum toxin serotype (such as serotype B). Another example of a modified botulinum toxin is a botulinum toxin coupled to a neurotransmitter, such as substance P.

“Mutation” means a structural modification of a naturally occurring protein or nucleic acid sequence. For example, in the case of nucleic acid mutations, a mutation can be a deletion, addition or substitution of one or more nucleotides in the DNA sequence. In the case of a protein sequence mutation, the mutation can be a deletion, addition or substitution of one or more amino acids in a protein sequence. For example, a specific amino acid comprising a protein sequence can be substituted for another amino acid, for example, an amino acid selected from a group which includes the amino acids alanine, asparagine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, proline, glutamine, arginine, serine, threonine, valine, tryptophan, tyrosine or any other natural or non-naturally occurring amino acid or chemically modified amino acids. Mutations to a protein sequence can be the result of mutations to DNA sequences that when transcribed, and the resulting mRNA translated, produce the mutated protein sequence. Mutations to a protein sequence can also be created by fusing a peptide sequence containing the desired mutation to a desired protein sequence.

“Patient” means a human or non-human subject receiving medical or veterinary care. Accordingly, the compositions as disclosed herein can be used in treating any animal, such as, for example, mammals, or the like.

“Peptide” and “polypeptide” refer to any polymer made up of a chain of amino acid residues linked by peptide bonds, regardless of its size. Although “protein” is often used in reference to relatively large polypeptides, and “peptide” is often used in reference to small polypeptides, usage of these terms in the art overlaps and varies. Thus, for simplicity, the term “polypeptide” will be used herein, although in some cases the art may refer to the same polymer as a “protein.” Unless otherwise indicated, the sequence for a polypeptide is given in the order from the amino terminus to the carboxyl terminus.

An amino acid sequence or a nucleotide sequence is “substantially the same as” or “substantially similar to” a reference sequence if the amino sequence or nucleotide sequence has at least 85% sequence identity with the reference sequence over a given comparison window. Thus, substantially similar sequences include those having, for example, at least 85% sequence identity, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity. Two sequences that are identical to each other are also substantially similar. The comparison window or the length of comparison sequence will generally be at least the length of the binding domain of a Botulinum toxin or binding domain of Botulinum toxin fragment, e.g., a fragment comprising about 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 150, 160, 180, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425 contiguous amino acids of the binding domain of a Botulinum toxin. Sequence identity is calculated based on the reference sequence, and algorithms for sequence analysis are known in the art. Thus, to determine percent sequence identity of two amino acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of one polypeptide for optimal alignment with the other polypeptide). The amino acid residues at corresponding acid positions are then compared. When a position in one sequence is occupied by the same amino acid residue as the corresponding position in the other sequence, then the molecules are identical at that position. The percent sequence identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., percent sequence identity=numbers of identical positions/total numbers of positions ×100). Percent sequence identity between two polypeptide sequences can be determined using the Vector NTI software package (Invitrogen Corp., Carlsbad, Calif.). A gap opening penalty of 10 and a gap extension penalty of 0.1 may be used for determining the percent identity of two polypeptides. All other parameters may be set at the default settings. Another software tool for determining sequence homology is The Basic Local Alignment Search Tool (BLAST) from National Center for Biotechnology Information (NCBI). For example, BLAST can compare protein sequences and calculate the percentage of identical or similar amino acid residues, homology, gaps, etc.

“Peripherally administering” or “peripheral administration” means subdermal, intradermal, transdermal, or subcutaneous administration, but excludes intramuscular administration. “Peripheral” means in a subdermal location and excludes visceral sites.

“Pharmaceutical composition” means a composition comprising an active pharmaceutical ingredient, such as, for example, a Clostridial toxin active ingredient such as a botulinum toxin, and at least one additional ingredient, such as, for example, a stabilizer or excipient or the like. A pharmaceutical composition is therefore a formulation which is suitable for diagnostic or therapeutic administration to a subject, such as a human patient. The pharmaceutical composition can be, for example, in a lyophilized or vacuum dried condition, a solution formed after reconstitution of the lyophilized or vacuum dried pharmaceutical composition, or as a solution or solid which does not require reconstitution.

“Pharmacologically acceptable excipient” is synonymous with “pharmacological excipient” or “excipient” and refers to any excipient that has substantially no long term or permanent detrimental effect when administered to mammal and encompasses compounds such as, e.g., stabilizing agent, a bulking agent, a cryo-protectant, a lyo-protectant, an additive, a vehicle, a carrier, a diluent, or an auxiliary. An excipient generally is mixed with an active ingredient, or permitted to dilute or enclose the active ingredient and can be a solid, semi-solid, or liquid agent. It is also envisioned that a pharmaceutical composition comprising a Clostridial toxin active ingredient can include one or more pharmaceutically acceptable excipients that facilitate processing of an active ingredient into pharmaceutically acceptable compositions. Insofar as any pharmacologically acceptable excipient is not incompatible with the Clostridial toxin active ingredient, its use in pharmaceutically acceptable compositions is contemplated. Non-limiting examples of pharmacologically acceptable excipients can be found in, e.g., Pharmaceutical Dosage Forms and Drug Delivery Systems (Howard C. Ansel et al., eds., Lippincott Williams & Wilkins Publishers, 7^(th) ed. 1999); Remington: The Science and Practice of Pharmacy (Alfonso R. Gennaro ed., Lippincott, Williams & Wilkins, 20th ed. 2000); Goodman & Gilman's The Pharmacological Basis of Therapeutics (Joel G. Hardman et al., eds., McGraw-Hill Professional, 10^(th) ed. 2001); and Handbook of Pharmaceutical Excipients (Raymond C. Rowe et al., APhA Publications, 4^(th) edition 2003), each of which is hereby incorporated by reference in its entirety.

The constituent ingredients of a pharmaceutical composition can be included in a single composition (that is, all the constituent ingredients, except for any required reconstitution fluid, are present at the time of initial compounding of the pharmaceutical composition) or as a two-component system, for example a vacuum-dried composition reconstituted with a reconstitution vehicle which can, for example, contain an ingredient not present in the initial compounding of the pharmaceutical composition. A two-component system can provide several benefits, including that of allowing incorporation of ingredients which are not sufficiently compatible for long-term shelf storage with the first component of the two-component system. For example, the reconstitution vehicle may include a preservative which provides sufficient protection against microbial growth for the use period, for example one-week of refrigerated storage, but is not present during the two-year freezer storage period during which time it might degrade the toxin. Other ingredients, which may not be compatible with a botulinum toxin or other ingredients for long periods of time, can be incorporated in this manner; that is, added in a second vehicle (e.g. in the reconstitution vehicle) at the approximate time of use. A pharmaceutical composition can also include preservative agents such as benzyl alcohol, benzoic acid, phenol, parabens and sorbic acid. Pharmaceutical compositions can include, for example, excipients, such as surface active agents; dispersing agents; inert diluents; granulating and disintegrating agents; binding agents; lubricating agents; preservatives; physiologically degradable compositions such as gelatin; aqueous vehicles and solvents; oily vehicles and solvents; suspending agents; dispersing or wetting agents; emulsifying agents, demulcents; buffers; salts; thickening agents; fillers; antioxidants; stabilizing agents; and pharmaceutically acceptable polymeric or hydrophobic materials and other ingredients known in the art and described, for example in Genaro, ed., 1985, Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., which is incorporated herein by reference.

“Polysaccharide” means a polymer of more than two saccharide molecule monomers. The monomers can be identical or different.

“Stabilizing agent”, “stabilization agent” or “stabilizer” means a substance that acts to stabilize a Clostridial toxin active ingredient such that the potency of the pharmaceutical composition is increased relative to an unstabilized composition.

“Stabilizers” can include excipients, and can include protein and non-protein molecules.

“Surfactant” refers to a natural or synthetic amphiphilic compound. A surfactant can be non-ionic, zwitterionic, or ionic. Non-limiting examples of surfactants include a poloxamer, a polysorbate, and combinations thereof.

“Therapeutic formulation” means a formulation can be used to treat and thereby alleviate a disorder or a disease, such as, for example, a disorder or a disease characterized by hyperactivity (i.e. seborrhea) of a sebaceous gland.

“Therapeutically effective concentration”, “therapeutically effective amount,” “effective amount,” “effective dose,” and “therapeutically effective dose” refer to the minimum dose of a Clostridial toxin active ingredient necessary to achieve the desired therapeutic effect and includes a dose sufficient to reduce a symptom associated with aliment being treated.

“Topical administration” excludes systemic administration of the neurotoxin. In other words, and unlike conventional therapeutic transdermal methods, topical administration of botulinum toxin does not result in significant amounts, such as the majority of, the neurotoxin passing into the circulatory system of the patient.

“Treating” means to alleviate (or to eliminate) at least one symptom of a condition or disorder, such as, for example, a fibrotic disorder, a disorder associated with the extracellular matrix, a disorder associated with connective, epithelial, or endothelial tissues, or the like, either temporarily or permanently. The term includes not only cure of a disease, but prevention of disease, control or even steps taken to mitigate a disease or disease symptoms. For instance, in reference to methods of treating a disorder, such as fibrosis, e.g., idiopathic pulmonary fibrosis (IPF) or cystic fibrosis (CF), the embodiment, generally includes the administration of a compound or composition which reduces the frequency of, or delays the onset of, symptoms of a medical condition (e.g., IPF or CF) in a subject relative to a subject not receiving the compound or composition. This can include reversing, reducing, or arresting the symptoms, clinical signs, and underlying pathology of a condition in a manner to improve or stabilize a subject's condition (e.g., regression of lung capacity).

In some embodiments, the therapeutic embodiments are carried out by contacting a tissue of a subject, e.g., endothelial tissue of the airways, with a delivery system. As defined herein, “contacting” means that the composition comprising the active ingredient (e.g., active pharmaceutical ingredient or API containing the polypeptide of the disclosure) is introduced into a sample containing a target, e.g., cell target, in a test tube, flask, tissue culture, chip, array, plate, microplate, capillary, or the like, and incubated at a temperature and time sufficient to permit binding of the peptide or the compound to the target. In the in vivo therapeutic context, “contacting” means that the polypeptide is introduced into a patient or a subject for the treatment of a disease of the disclosure, e.g., IPF, CF, and the polypeptide or the compound is allowed to come in contact with the patient's target tissue, e.g., epithelial tissue, in vivo.

In some embodiments, the therapeutic applications are carried out by administering the compositions or kits to a subject, e.g., a patient suffering from a disease such as IPF or CF. The term “administering” means applying as a remedy, such as by the placement of a drug in a manner in which such drug would be received and be effective in carrying out its intended purpose.

As used herein, the term “disease of the disclosure” or “disorder of the disclosure” includes one of the following classes of diseases: extracellular matrix disease; connective or epithelial tissue disease; endothelial diseases; and fibrotic diseases; preferably fibrotic diseases; and particularly fibrotic disease of the ECM, connective or epithelial tissue, or endothelial tissue.

As used herein, the term “extracellular matrix disease” or “disease associated with extracellular matrix (ECM)” refers to a condition, disorder or disease, associated with the ECM or one or more components thereof. The ECM provides structural support to cells in addition to being involved in other biological functions including, but not limited to, intracellular communication. Components of the ECM include, but are not limited to, proteoglycans (e.g., heparan sulfate, chondroitin sulfate, and keratin sulfate), non-proteoglycan polysaccharaides (e.g., hyaluronic acid), fibers, collagen, elastin, fibronectin and laminin. The extracellular matrix also serves as a depot for signaling molecules such as growth factors and cytokines. Extracellular matrix diseases include diseases associated with the dysregulation of one or more functions of the ECM (e.g., dysregulated intracellular communication and/or movement) or dysreguation of one or more components of the ECM (e.g., increased or decreased activity and/or production of one or more components of the ECM). Extracellular matrix diseases also include diseases associated with altered degradation and remodeling of the ECM and diseases associated with altered (e.g., increased or decreased) accumulation of agents, e.g., immunocomplexes and other immune products, in the ECM. Extracellular matrix diseases include, but are not limited to, atherosclerosis, cancer, amyloid diseases, glomerular diseases, mesangial diseases, inflammatory conditions, and developmental disorders. Cancers include acute lymphoblastic leukemia, dermatofibromas, breast cancer, breast carcinoma, glioma and glioblastoma, rhabdomyosarcoma and fibrosarcoma, desmoplasia, angiolipoma, angioleiomyoma, desmoplastic cancers, and prostate, ovarian, colorectal, pancreatic, gastrointestinal, and liver cancer and other tumor growth and metastases.

As used herein, the term “disease associated with connective tissue” or “connective tissue disease” refers to a condition, disorder or disease, associated with the connective tissues or one or more components thereof. The term “connective tissue” refers to tissue that supports, connects, or separates different types of tissues and organs of the body. It is one of the four general classes of animal tissues. Connective tissue is found everywhere including in the central nervous system. It is located in between other tissues. All connective tissue has three main components: ground substances, fibers and cells. Preferred connective tissues are tissues comprising collagenous fibers. Representative examples of connective tissue diseases include systemic lupus erythematosus (SLE), rheumatoid arthritis, systemic sclerosis, {umlaut over (S)}jogren's syndrome, mixed connective tissue disease, eosinophilia infectiosa, polymyositis, dermatomysistis, periarteriitis nodosa, Wegener-granulomatose, CREST-syndrome and SHARP-syndrome; preferably systemic lupus erythematosus (SLE), rheumatoid arthritis, systemic sclerosis, or Sjörgen's syndrome.

As used herein, the term “disease associated with epithelial tissue” or “epithelial tissue disease” refers to a condition, disorder or disease, associated with the connective tissues or one or more components thereof. The term “epithelial tissue” refers to tissue that lines the outer surfaces of organs and blood vessels throughout the body, as well as the inner surfaces of cavities in many internal organs. It is one of the four general classes of animal tissues. Representative examples of epithelial or connective tissue diseases include eczema, atopic dermatitis, psoriasis, dermal scars (hypertrophic, keloid, atropic (for example with acne), stretch marks, and umbilical), wound scars, surgical scars (for example after cesarean section, abdominoplasty, breast augmentation, abdominal surgery, face lift, or injection of fillers), postoperative intraperitoneal adhesions, fibroma, liver fibrosis, cirrhosis, pancreatitis, benign prostatic hyperplasia, fibrotic bladder, interstitial cystitis, pulmonary fibrosis, kidney fibrosis, glomerulosclerosis, atrial fibrosis, endomyocardial fibrosis, glial scar, arterial stiffness, arthrofibrosis, cystic fibrosis, non-cystic fibrosis, crohn's disease, dupuytren's contracture, mediastinal fibrosis, myelofibrosis, peyronie's disease, nephrogenic systemic fibrosis, progressive massive fibrosis, retroperitoneal fibrosis, adhesive capsulitis, mixed connective tissue disease, ocular fibrosis, osteoarthritis, scleroderma, and asthma, and the like.

As used herein, the term “disease associated with endothelium” or “endothelial disease” refers to a condition, disorder or disease, associated with the endothelial tissues or one or more components thereof. The term “endothelium” refers to a thin layer of simple, or single-layered, squamous cells called endothelial cells, which line the interior surface of blood vessels and lymphatic vessels, forming an interface between circulating blood or lymph in the lumen and the rest of the vessel wall. In some embodiments, the endothelial disorder is selected from the group consisting of Alzheimer's disease, amyotrophic lateral sclerosis, diabetic neuropathy, stroke, atherosclerosis, diabetes, restenosis, coronary artery disease, peripheral vascular disease, vascular leak, vasculitis, vasculitidis, Wegner's disease, gastric or oral ulcerations, cirrhosis, hepatorenal syndrome, Crohn's disease, hair loss, skin purpura, telangiectasia, venous lake formation, delayed wound healing, pre-eclampsia, sepsis, ischemia-reperfusion injury, hypertension, chronic or acute infection, menorrhagia, neonatal respiratory distress, pulmonary fibrosis, emphysema, nephropathy, glomerulonephritis, sclerodoma, and vascular abnormalities. In some embodiments, endothelial diseases are characterized by insufficient angiogenesis, hypertension, vasoconstriction, vascular leak, altered vasomotor tone, hypercoagulation, anti-inflammatory properties, and poor endothelial cell health.

As used herein, the term “fibrotic disorder” or “fibrotic disease” refers to a medical condition featuring progressive and/or excessive fibrosis, which is generally characterized by thickening or scarring of connective tissues, e.g., due to injury. In some embodiments, fibrotic disease is characterized by excessive deposition of extracellular matrix occurs in and around inflamed or damaged tissue. In certain embodiments, a fibrotic disorder or disease is associated with the persistent presence of myofibroblasts in and around fibrotic foci or lesions. Excessive and persistent fibrosis can progressively remodel and destroy normal tissue, which may lead to dysfunction and failure of affected organs, and ultimately death.

In some embodiments, the fibrotic disorder may be characterized by “transdifferentiation”, which refers to the direct conversion of one cell type into another. Particularly, fibrotic disorder or fibrotic disease includes fibrosis of ECM, connective tissues, endothelial tissues, or a combination thereof. In some embodiments, the term fibrotic disease excludes fibrosis triggered by normal wound healing.

Non-limiting examples of fibrotic disorders or fibrotic diseases include dermal scars, wound scars, surgical scars, postoperative intraperitoneal adhesions, fibroma, liver fibrosis, cirrhosis, pancreatitis, benign prostatic hyperplasia, breast fibrosis, fibrotic bladder, interstitial cystitis, pulmonary fibrosis (e.g., idiopathic pulmonary fibrosis), vascular fibrosis (e.g., atherosclerosis, stenosis, restenosis), kidney fibrosis, glomerulosclerosis, atrial fibrosis, cardiac fibrosis, endomyocardial fibrosis, glial scar, arterial stiffness, arthrofibrosis, mediastinal fibrosis, myelofibrosis, retroperitoneal fibrosis, progressive massive fibrosis (e.g., lungs), chronic kidney disease (CKD), nephrogenic systemic fibrosis, Crohn's disease, hypertrophic scarring, keloid, scleroderma, systemic sclerosis (e.g., skin, lungs), athrofibrosis (e.g., knee, shoulder, other joints), Peyronie's disease, Dupuytren's contracture, adhesive capsulitis, organ transplant associated fibrosis, ischemia associated fibrosis, cystic fibrosis, non-cystic fibrosis, mixed connective tissue disease, ocular fibrosis, osteoarthritis, scleroderma, asthma or the like. Preferably, the fibrotic disorder is fibrotic breast disease; keloid scar; liver fibrosis; fibrotic bladder; hyperplasia of fibrous tissue, e.g., benign prostatic hyperplasia; pancreatitis; adhesion, e.g., intra-abdominal adhesion; scarring, e.g., hypertrophic scars or a combination thereof.

As used herein, the term “variant” refers to a biomolecule (e.g., polypeptide or nucleic acid) whose sequence that differs from that of a parent sequence by virtue of at least one modification or amino acid (or nucleic acid) substitution. Accordingly, variant polypeptides comprise at least one modification or substitution of an amino acid residue. Types of modifications that give raise to variant polypeptides include, e.g., addition, deletion, substitution, transposition, etc. of one or more amino acid residues.

Polypeptides and Compositions Comprising the Polypeptides

As mentioned above, in one aspect, a polypeptide is provided that comprises an amino acid sequence substantially identical to an amino acid sequence in a binding domain, of a Clostridial toxin. In one embodiment, the Clostridial toxin is a botulinum toxin. In some embodiments, the polypeptide comprises an amino acid sequence substantially identical to an amino acid sequence in the full-length of a botulinum toxin which is devoid of toxicity. In one embodiment, the polypeptide comprises an amino acid sequence substantially identical to the amino acid sequence of the full-length of a botulinum toxin which is devoid of toxicity. In some embodiments, the polypeptide comprises an amino acid sequence substantially identical to an amino acid sequence in the heavy chain of the botulinum toxin. In some embodiments, the polypeptide comprises an amino acid sequence substantially identical to an amino acid sequence in the carboxyl or C-terminal segment of the heavy chain of botulinum toxin (H_(C)). In one embodiment, the polypeptide comprises an amino acid sequence substantially identical to an amino acid sequence of the binding domain of the botulinum toxin. In one embodiment, the polypeptide comprises an amino acid sequence identical to the amino acid sequence of the binding domain of the botulinum toxin. In another embodiment, the polypeptide comprises an amino acid sequence substantially identical to an amino acid sequence in the amino or N-terminal half of the binding domain of the botulinum toxin (H_(CN)). In one embodiment, the polypeptide comprises an amino acid sequence identical to the N-terminal half of the binding domain of the botulinum toxin. In one embodiment, the botulinum toxin is a BoNT/A. In another embodiment, the botulinum toxin is a mosaic toxin. In one embodiment, the botulinum toxin is BoNT/DC. In alternative embodiments, the botulinum toxin is not a BoNT/A.

In another aspect, a polypeptide comprises a sequence of amino acids having at least 90% sequence identity to a binding domain of a botulinum toxin. In one embodiment, the molecular weight of the polypeptide is between about 1 kDa to about 90 kD, or between about 20 kDa to about 60 kD, or between about 22 kDa to about 50 kD.

The amino acid sequences of the botulinum toxin serotypes and subtypes are known and Table 1 gives approximate boundary regions for translocation, endopeptidase and binding domains, as well as exemplary GENBANK/UNIPROT accession number(s) thereof. Representative serotypes of BoNT toxins, e.g., type A, type B, type C₁, type D, type E, type F, type G, including, related members are also disclosed in U.S. Pat. Nos. 7,892,565 and 8,486,422. A potential eighth type (“type H”) was described in Dover et al., J Infect Dis., 209(2):192-202, 2014 (PMID: 24106295). Recent reports have variously described this novel neurotoxin as BoNT/H, BoNT/FA or BoNT/HA. See, Maslanka et al., J Infect Dis., 213(3): 379-385, 2016; Peck et al., Toxins (Review), 9(1): 38, 2017. Mosaic botulinum toxins are also known, such as BoNT/DC and BoNT/CD.

TABLE 1 Toxin ACCESSION # LC HN HC BoNT/A P0DPI1 M1-K448 A449-K871 N872-L1296 BoNT/B P10844 M1-K441 A442-S858 E859-E1291 BoNT/C₁ P18640 M1-K449 T450-N866 N867-E1291 BoNT/D P19321 M1-R445 D446-N862 S863-E1276 BoNT/E Q00496 M1-R422 K423-K845 R846-K1252 BoNT/F P30996 M1-K439 A440-K864 K865-E1274 BoNT/G Q60393 M1-K446 S447-S863 N864-E1297 BoNT/H KGO15617 M1-K434 N435-S843 Y844-L1288 BoNT/DC ABP48747 M1-K500 V501-K831 V832-E1285 TeNT P04958 M1-A457 S458-V879 I880-D1315 BaNT Q45851 M1-K431 N432-I857 I858-E1268 BuNT P30995 M1-R422 K423-I847 K848-K1251 eBoNT/J A0A242DI27 M1-Q432 R433-I860 D861-D1279

In one embodiment, the Clostridial toxin is derived from BoNT/A. In an alternate embodiment, the Clostridial toxin is not derived from BoNT/A, e.g., the Clostridial toxin is a Clostridial toxin derived from BoNT/B, BoNT/C₁, BoNT/D, BoNT/E, BoNT/F, BoNT/G, BoNT/H, BoNT/DC, BoNT/CD, BoNT/X, eBoNT/J, TeNT, BaNT, BuNT, or a combination thereof.

In another embodiment, Clostridial toxins are derived from various subtypes of Clostridial toxins. As used herein the term “subtype” may refer to any of two or more functionally similar proteins that have identical or similar amino acid sequences and are either encoded by different genes, or by RNA transcripts from the same gene which have had different exons removed. “Subtype” also may refer to any of the sequences encoding such proteins, including mature and immature sequences. Thus, “subtype” includes the genes encoding one or more of the aforementioned Clostridial toxins, as well as the protein products of the genes, unless stated or otherwise understood by context to refer to only one or the other. At least 40 unique BoNTs, often called subtypes, have been identified by DNA sequencing; some have an impact on BoNT function (Rossetto et al., Nature Reviews Microbiol., 12, 535-49, 2014). For instance, molecular studies have provided evidence for cross-reactive serological observations of a single BoNT containing structural components of BoNT serotypes C and D. See, Arndt et al. (J Mol Biol., 362(4):733-42, 2006) and Hill et al., (J Bacteriol., 189:818-32, 2007). In some instances, sequences of BoNT/F were found to be particularly variable (Raphael et al., Appl Environ Microbiol., 76(14):4805-12, 2012), leading to functional diversity with regard to cleavage of synaptic vesicle membrane proteins such as VAMP-2 (Kalb et al., Anal Chem., 86:3254-62, 2014).

In one embodiment, the Clostridial toxin is derived from various BoNT/A subtypes, such as, e.g., A1, A2, A3, A4, A5, A6, A7, A9, A10; BoNT/B subtypes, such as, e.g., B1, B2, B3, B4, B5, B6, B7, B8, Bnp, and Bbv; BoNT/C subtypes, such as, e.g., C and CD; BoNT/D subtypes, such as, e.g., D and DC; BoNT/E subtypes, such as, e.g., E1, E2, E3, E4, E5, E6, E7, E8, E9; BoNT/F subtypes, such as, e.g., F1, F2, F3, F4, F5, F6, F7; and BoNT/G subtypes, such as, e.g., subtype G. BoNT subtypes include chimeric BoNTs e.g., BoNT/DC, BoNT/CD, BoNT/FA, etc. See, Rossetto et al., Nature Reviews Microbiol., 12, 535-49, 2014; Montecucco et al., M Bio 6:e02131-14, 2015; U.S. Pat. Nos. 8,841,111 and 8,697,413. Analysis of sequence alignment, for example, performed via software such as VECTOR NTI (Thermo Fisher, Carlsbad, Calif.), reveals a high degree of sequence identity between the individual BoNT/A subtypes. For example, in one embodiment, there is about 78.9% (e.g., between 75%-80%, depending on the alignment software) overall sequence identity and an even greater sequence identity of about 99.3% (e.g., between 95% and 99.9%, depending on the alignment software) at the consensus positions.

In one embodiment, the disclosure relates to homologs of Clostridial toxins. The term “homolog” means a protein in a group of proteins that perform the same biological function, e.g., proteins that belong to the same Clostridial toxin family and that provide a common activity Homologs are generally expressed by homologous genes. With reference to homologous genes, homologs include orthologs, e.g., genes expressed in different species that evolved from a common ancestral gene by speciation and encode proteins retain the same function, but do not include paralogs, e.g., genes that are related by duplication but have evolved to encode proteins with different functions. Homologous genes include naturally occurring alleles and artificially-created variants. Degeneracy of the genetic code provides the possibility to substitute at least one base of the protein encoding sequence of a gene with a different base without causing the amino acid sequence of the polypeptide produced from the gene to be changed. When optimally aligned, homolog proteins have typically at least about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 92%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5% or greater % identity or similarity compared to a subject protein, e.g., BoNT/A1 or a fragment thereof comprising the H_(C) domain, particularly the H_(CN) domain, especially the N-terminal half of the H_(CN) domain. In another aspect of the disclosure homolog proteins have an amino acid sequences that have at least about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90% or greater % identity or similarity to the polypeptides of SEQ ID NO: 1, SEQ ID NO: 19, SEQ ID NOs: 3-5, SEQ ID NO: 6, SEQ ID NO: 20, SEQ ID Nos: 7-10, SEQ ID NO: 11, SEQ ID NO: 21, and SEQ ID NOs: 12-18.

In one embodiment, homologous Clostridial toxins are grouped on the basis of percent homology, which is defined as the percent of either identical or similar residues (consensus) within a protein sequence relative to a reference protein sequence, divided by the length of the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence homology. Consensus substitutions are those substitutions that allow an amino acid to be substituted with a similar amino acid. Amino acids can be similar in several characteristics, for example, size, shape, hydrophobicity, hydrophilicity, charge, isoelectric point, polarity, aromaticity, etc. Alignment for purposes of determining percent amino acid sequence homology can be achieved in various ways that are within the ordinary skill of those persons of skill in the art. In some cases, amino acid sequences can be aligned using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN (DNASTAR) software. In one embodiment, the percent homology is computed using Basic Local Alignment Search Tool (BLAST) available via the NCBI, which conducts a pairwise alignment and provides a raw score using the matrix of residue substitution. Herein, the bit score computed by BLAST is a normalized score which considered the sequence length and gap size. As is understood in bioinformatics, a score of 283 bits means to find a better alignment than what is presented, the search would have to encompass a space of 2²⁸³ (or 2×10⁸⁵) units (e.g., amino acids or nucleic acids). Thus, the higher the bit score, the more highly significant the match. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. Sequence homology is then calculated relative to the longer sequence, i.e., even if a shorter sequence shows 100% sequence identity with a portion of a longer sequence, the overall sequence identity will be less than 100%.

Under an alternate embodiment, the variant Clostridial toxin may comprise a sequence which shares at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater degree of identity to one or more of the aforementioned Clostridial toxins. For example, in one embodiment, variant Clostridial toxins may comprise at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater, e.g., about 99.9%, sequence identity to the amino acid sequence set forth in UNIPROT Accession Nos. P0DPI1 (e.g., type A); P10844 (e.g., type B); P18640 (e.g., type C₁); P19321 (e.g., type D); Q00496 (GENBANK #CAA44558) (e.g., type E); P30996 (e.g., type F); Q60393 (e.g., type G); GENBANK ID: KG015617 (UNIPARC ID: 00052C1529) for type H; GENBANK ID: BAQ12790 (UNIPARC ID: 0005822796) for type X; UNIPROT Accession Nos. P04958 (e.g., TeNT); Q45851 (e.g., BaNT); or P30995 (e.g., BuNT).

In another embodiment, variant Clostridial toxins may comprise at least about 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater, e.g., about 99.9%, sequence identity to the nucleic acid or amino acid sequence set forth in (a) GENBANK Accession Nos. AF488749 (nucleic acid) and AAQ06331 (protein) for toxins derived from BoNT/A; (b) GENBANK Accession Nos. AB232927 (nucleic acid) or BAE48264 (protein) for toxins derived from BoNT/B; (c) GENBANK #CAA47060 (nucleic acid); UNIPROT #P18640 (protein) for toxins derived from BoNT/C1; (d) GENBANK #X54254 (nucleic acid); UNIPROT #P19321 (protein) for toxins derived from BoNT/D; (e) GENBANK #EF378947 (nucleic acid); ABP48747 (protein) for toxins derived from BoNT/DC; (f) GENBANK #JX424539 (nucleic acid); AFV91344 (protein) for toxins derived from BoNT/E; (g) GENBANK #ABS41202 (protein); DNA (e.g., cDNA) encoding ABS41202 for toxins derived from BoNT/F; (h) GENBANK #X74162 (nucleic acid); UNIPROT #Q60393 (protein) for toxins derived from BoNT/G; (i) GENBANK ID: BAQ12790 (protein) for toxins derived from BoNT/X; or (j) GENBANK #OTO22244; UNIPROT #A0A242DI27 (protein) for toxins derived from eBoNT/J.

In yet another embodiment, variant Clostridial toxins may comprise at least about 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater, e.g., about 99.9%, sequence identity to the nucleic acid or amino acid sequence set forth in (1) GENBANK #AB443580 (nucleic acid); BAH03558 (protein) for a toxin derived from BoNT/A2 Chiba strain; (b) GENBANK #X73423 (nucleic acid); CAA51824 (protein)) for toxins derived from Kyoto-F strain; (c) GENBANK #DQ185900 (nucleic acid); ABA29017 (protein) for toxins derived from BoNT/A3 Loch Maree strain; (d) (GENBANK #EU341307 (nucleic acid); ABY56338 (protein)) for toxins derived from BoNT/A4 657Ba strain; (e) (GENBANK #HM153705 (nucleic acid); ADJ68235 (protein) for toxins derived from BoNT/A5A 661222 strain; (f) GENBANK #FJ981696 (nucleic acid); ACW83602 (protein) for toxins derived from BoNT/A6 CDCl₄1370 strain; GENBANK #JQ954969 (nucleic acid); AFV13854 (protein) for toxins derived from BoNT/A7 2008-148 strain.

In some embodiments, the variant Clostridial toxins include mutant Clostridial toxins or a fragment thereof. Typically, mutant Clostridial toxins comprise at least 1, 2, 3, 4, 5, or more, e.g., up to 10 mutations in a core Clostridial toxin polypeptide sequence or a fragment thereof, e.g., full-length BoNT/A sequence or the H_(C) fragment of SEQ ID NO: 1. Representative examples of such mutant Clostridial toxins include, e.g., a first BoNT/A mutant comprising W1266L and Y1267S or a fragment thereof (e.g., a H_(C) fragment of the BoNT/A mutant comprising W391L and Y392S in SEQ ID NO: 1; mutant sequence set forth in SEQ ID NO: 26) or a second BoNT/A mutant comprising T1145A and T1146A or a fragment thereof (e.g., a H_(C) fragment of the BoNT/A mutant comprising T270A and T271A in SEQ ID NO: 1; mutant sequence set forth in SEQ ID NO: 25) or a third BoNT/A mutant comprising G1292R or a fragment thereof (e.g., a H_(C) fragment of the BoNT/A mutant comprising G417R in SEQ ID NO: 1; mutant sequence set forth in SEQ ID NO: 27). In some embodiments, the mutant polypeptides or fragments thereof have modulated, e.g., diminished, in vitro or in vivo activity compared to the non-muted Clostridial toxins or fragments thereof and the mutant polypeptides may therefore be used to modulate the pharmacological properties of the non-muted Clostridial toxins or fragments thereof.

Any of a variety of sequence alignment methods can be used to determine percent identity, including, without limitation, global methods, local methods and hybrid methods, such as, e.g., segment approach methods. Protocols to determine percent identity are routine procedures within the scope of one skilled in the art and from the teaching herein.

In one embodiment, the variant Clostridial toxin comprises one or more amino acid substitutions, which are selected so as to preserve activity of the variant Clostridial toxin. Residues that are semi-conserved may tolerate changes that preserve charge, polarity, and/or size. For example, a variant Clostridial toxin comprising the amino acid sequence set forth in the aforementioned accessioned UNIPROT and/or GENBANK sequences may have one or more substitutions, wherein the substituted amino acid may be any one of the known 20 amino acids, wherein the variant Clostridial toxin maintains its activity, e.g., cell-binding activity or cell-signaling activity or a combination thereof (see Examples section). Preferably, the amino acids are substituted in a conserved or semi-conserved manner. Exemplary types of conserved amino acid substitutions include, e.g., a substitution of a non-polar (hydrophobic) residue for another non-polar (hydrophobic) residue such as I, V, L or M for one another, a substitution of one polar (hydrophilic), non-charged residue for another polar, non-charged residue such as Q for N, G for S, or vice versa, or a substitution of a charged residue for another similarly charged residue such as K, R or H for one another, or D for E or vice versa. On the other hand, non-conservative substitutions include the substitution of a non-polar (hydrophobic) residue such as I, V, L, A, M for a polar (hydrophilic) residue such as C, Q, D, K and/or vice versa. In one embodiment, the term “conserved substitution” indicates an amino acid substitution within one of the following “strong” groups: STA, NEQK, NHQK, NDEQ, QHRK, MILV, MILF, HY, and/or FYW; and the term “semi-conserved substitution” indicates an amino acid substitution within one of the following “weak” groups: CSA, ATV, SAG, STNK, STPA, SGND, SNDEQK, NDEQHK, NEQHRK, FVLIM, and/or HFY. Methods of making conserved or semi-conserved amino acid substitutions are known in the art, e.g., Risler et al., J. Mol. Biol., 204:1019-1029, 1988; Niefind et al., J. Mol. Biol. 219:481-497, 1991; and Overington et al. Protein Science, 1:216-226, 1992. Exemplary types of conserved and semi-conserved amino acid substitutions in the core Clostridial toxin sequence (e.g., BoNT/A sequence) are observable via a CLUSTAL multiple sequence alignment, wherein the asterisk (*) indicates identity, the semicolon (:) indicates conserved substitution and the period (.) indicates a semi-conserved substitution.

Clostridial toxin variants of the present disclosure may be a naturally-occurring variant or a non-naturally-occurring variant. As used herein, the term “naturally occurring Clostridial toxin variant” refers to any Clostridial toxin produced without the aid of any human manipulation, including, without limitation, Clostridial toxin isoforms produced from alternatively-spliced transcripts, Clostridial toxin isoforms produced by spontaneous mutation and Clostridial toxin subtypes. As used herein, the term “non-naturally occurring Clostridial toxin variant” refers to any Clostridial toxin produced with the aid of human manipulation, including, without limitation, Clostridial toxins produced by genetic engineering using random mutagenesis or rational design and Clostridial toxins produced by chemical synthesis. Non-limiting examples of non-naturally occurring Clostridial toxin variants include, e.g., conservative Clostridial toxin variants, non-conservative Clostridial toxin variants, and active Clostridial toxin fragments. Non-natural Clostridial toxins further include natural Clostridial toxins that have been post-translationally modified, e.g., via addition of chemical moieties, tags, ligands, and the like.

As used herein, the term “conservative Clostridial toxin variant” refers to a Clostridial toxin that has at least one amino acid substituted by another amino acid or an amino acid analog that has at least one property similar to that of the original amino acid from the reference Clostridial toxin sequence (see, e.g., Table 1). Examples of such properties include, without limitation, similar size, topography, charge, hydrophobicity, hydrophilicity, lipophilicity, covalent-bonding capacity, hydrogen-bonding capacity, a physicochemical property, of the like, or any combination thereof. A conservative Clostridial toxin variant can function in substantially the same manner as the reference Clostridial toxin on which the conservative Clostridial toxin variant is based, and can be substituted for the reference Clostridial toxin in any aspect of the present specification. A conservative Clostridial toxin variant may substitute 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 75, 100, 200, 300, 400, or 500 or more amino acids from the reference Clostridial toxin on which the conservative Clostridial toxin variant is based. A conservative Clostridial toxin variant can also substitute at least 5, 10, 15, 20, or 25 contiguous amino acids from the reference Clostridial toxin on which the conservative Clostridial toxin variant is based. Non-limiting examples of a conservative Clostridial toxin variant include, e.g., conservative BoNT/A variants, conservative BoNT/B variants, conservative BoNT/C₁ variants, conservative BoNT/D variants, conservative BoNT/E variants, conservative BoNT/F variants, conservative BoNT/G variants, conservative BoNT/H variants, conservative BoNT/X variants, conservative eBoNT/J variants, conservative TeNT variants, conservative BaNT variants and conservative BuNT variants.

As used herein, the term “non-conservative Clostridial toxin variant” refers to a Clostridial toxin in which (a) at least one amino acid is deleted from the reference Clostridial toxin on which the non-conservative Clostridial toxin variant is based; (b) at least one amino acid added to the reference Clostridial toxin on which the non-conservative Clostridial toxin is based; or (c) at least one amino acid is substituted by another amino acid or an amino acid analog that does not share any property similar to that of the original amino acid from the reference Clostridial toxin sequence (see, e.g., Table 1). A non-conservative Clostridial toxin variant can function in substantially the same manner as the reference Clostridial toxin on which the non-conservative Clostridial toxin variant is based, and can be substituted for the reference Clostridial toxin in any aspect of the present specification. A non-conservative Clostridial toxin variant can delete one or more amino acids, two or more amino acids, three or more amino acids, four or more amino acids, five or more amino acids, and ten or more amino acids from the reference Clostridial toxin on which the non-conservative Clostridial toxin variant is based. A non-conservative Clostridial toxin variant can add one or more amino acids, two or more amino acids, three or more amino acids, four or more amino acids, five or more amino acids, and ten or more amino acids to the reference Clostridial toxin on which the non-conservative Clostridial toxin variant is based. A non-conservative Clostridial toxin variant may substitute 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 75, 100, 200, 300, 400, or 500 or more amino acids from the reference Clostridial toxin on which the non-conservative Clostridial toxin variant is based. A non-conservative Clostridial toxin variant can also substitute at least 5, 10, 15, 20, or 25 contiguous amino acids from the reference Clostridial toxin on which the non-conservative Clostridial toxin variant is based. Non-limiting examples of a non-conservative Clostridial toxin variant include, e.g., non-conservative BoNT/A variants, non-conservative BoNT/B variants, non-conservative BoNT/C₁ variants, non-conservative BoNT/D variants, non-conservative BoNT/E variants, non-conservative BoNT/F variants, non-conservative BoNT/G variants, non-conservative BoNT/H variants, non-conservative BoNT/X variants, non-conservative eBoNT/J variants, non-conservative TeNT variants, non-conservative BaNT variants and non-conservative BuNT variants. It is also envisioned that any of a variety of Clostridial toxin fragments can be useful in aspects of the present specification with the proviso that these active fragments can execute the overall cellular mechanism whereby a Clostridial toxin binds to a binding partner, e.g., synaptic vesicle glycoprotein. Thus, aspects of this embodiment can include Clostridial toxin fragments having a length of, e.g., at least 600, 700, 800, 900, 1000, 1100, or at least 1200 amino acids. Other aspects of this embodiment, can include Clostridial toxin fragments having a length of, e.g., at most 600, 700, 800, 900, 1000, 1100, or at most 1200 amino acids.

Embodiments of the disclosure further relate to variant Clostridial toxin polypeptides. As used herein, the term “polypeptide” includes a molecule comprising a linear chain or branched amino acids, peptidomimetics, as well as pharmaceutically acceptable salts thereof. Typically, a polypeptide comprises a plurality of amino acid residues, e.g., 2, 5, 10, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, or more amino acid residues which are bonded to each other via covalent bonds, e.g., a peptide bond. “Amino acid residue” means the individual amino acid units incorporated into the polypeptides of the disclosure. As used herein, the term “amino acid” means a naturally occurring or synthetic amino acid, as well as amino acid analogs, stereoisomers, and amino acid mimetics that function similarly to the naturally occurring amino acids. Included by this definition are natural amino acids such as: (1) histidine (His; H) (2) isoleucine (Ile; I) (3) leucine (Leu; L) (4) Lysine (Lys; K) (5) methionine (Met; M) (6) phenylalanine (Phe; F) (7) threonine (Thr; T) (8) tryptophan (Trp; W) (9) valine (Val; V) (10) arginine (Arg; R) (11) cysteine (Cys; C) (12) glutamine (Gln; Q) (13) glycine (Gly; G) (14) proline (Pro; P) (15) serine (Ser; S) (16) tyrosine (Tyr; Y) (17) alanine (Ala; A) (18) asparagine (Asn; N) (19) aspartic acid (Asp; D) (20) glutamic acid (Glu; E) (21) selenocysteine (Sec; U); including unnatural amino acids: (a) citrulline (Cit); (b) cystine; (c) gama-amino butyric acid (GABA); (d) ornithine (Orn); (f) theanine; (g) homocysteine (Hey); (h) thyroxine (Thx); and amino acid derivatives such as betaine; carnitine; carnosine creatine; hydroxytryptophan; hydroxyproline (Hyp); N-acetyl cysteine; S-Adenosyl methionine (SAM-e); taurine; tyramine.

In one embodiment, the Clostridial toxin comprises derivatives of parent Clostridial toxins, e.g., derivatives of the amino acid sequence set forth in UNIPROT Accession Nos. P0DPI1 (e.g., type A); P10844 (e.g., type B); P18640 (e.g., type C₁); P19321 (e.g., type D); Q00496 (GENBANK #CAA44558) (e.g., type E); P30996 (e.g., type F); Q60393 (e.g., type G); GENBANK ID: KG015617 (UNIPARC ID: 00052C1529) for type H; GENBANK ID: BAQ12790 (UNIPARC ID: 0005822796) for type X; UNIPROT Accession Nos. P04958 (e.g., TeNT); Q45851 (e.g., BaNT); or P30995 (e.g., BuNT). As used herein, the term “derivative” includes salts, amides, esters, acids, bases, solvates, hydrates, polymorphs or prodrugs of the individual amino acids or the aforementioned polypeptides, including fragments thereof. Such derivatives may be readily prepared by those of skill in this art using known methods for such derivatization. The derivatives suitable for use in the methods described herein may be administered to animals or humans without substantial toxic effects and either are biologically active or are prodrugs.

In one example, the derivatives comprise salts of the amino acids or the toxin polypeptides. The term “salt” includes salts derived from any suitable of organic and inorganic counter ions well known in the art and include, by way of example, hydrochloric acid salt or a hydrobromic acid salt or an alkaline or an acidic salt of the aforementioned amino acids.

If desired, the derivative can, in addition or alternatively, be a solvent addition forms, e.g., a solvate or alcoholate. Solvates contain either stoichiometric or non-stoichiometric amounts of a solvent, and may be formed during the process of crystallization with acceptable solvents such as water, ethanol, and the like. Hydrates are formed when the solvent is water; alcoholates are formed when the solvent is alcohol. Solvates of compounds described herein can be conveniently prepared or formed using routine techniques.

In another embodiment, disclosed herein are polymorphs of Clostridial toxins. Polymorphs refer to alternate crystal forms of the Clostridial toxins described herein. Polymorphic purity of protein (or a polypeptide) samples can be checked using techniques such as powder X-ray diffraction, IR/Raman spectroscopy, and utilizing the differences in their optical properties in some cases (Thomas et al., Chemical Communications, 48: 10559-10561 (2012)).

The derivative can further comprise amides or esters of the amino acids and/or isomers (e.g., tautomers or stereoisomers) of the amino acids, as desired.

Domains

Embodiments of the disclosure further relate to domains and sites present in the aforementioned Clostridial toxins, e.g., the translocation domain (H_(N)), the endopeptidase domain, or the cell-binding domain, including, including sub-domains thereof, e.g., H_(CN) sub-domain or H_(CC) subdomain. In one embodiment, the disclosure relates to a polypeptide comprising a cell-binding site located within one or more domains or subdomains in the Clostridial toxin. Such polypeptides can comprise, e.g., one or more amino acid motifs of Clostridial toxin cell-binding domains. Boundary regions for each domain and subdomain found in exemplary Clostridial toxins are disclosed for example in U.S. Pat. No. 8,697,413, incorporated entirely herein by reference. Boundary regions of various domains may be approximated using art-known bioinformatics tools (e.g., INTERPRO or PROSITE). Accordingly, the boundary regions as disclosed in the '413 patent are not absolute and minor variations, e.g., a difference of 1, 2, 3, 4, 5, 7, 10, 15, 20, or more amino acids, each representing a change of less than about 5%, about 4%, about 3%, about 2%, about 1% or a smaller % in the length and/or the molecular weight of the individual domains, is permissible, as disclosed for example in U.S. Pat. Nos. 8,841,111 and 8,512,992, incorporated entirely herein by reference.

The binding activity of the Clostridial toxin or fragments thereof may be assayed using routine techniques, e.g., radio-labeled assay, competitive binding assay, in vitro binding assays such as BIAcore Surface Plasmon (SPR) technology, receptor phosphorylation, dimerization or signaling assay, etc. A representative assay for assessing the biological activity of a polypeptide corresponding to a Clostridial toxin fragment, comprising, e.g., assessing modulation in gene expression mediated by Clostridial toxin variants, comprising, detecting a level of plurality of genes described in the Examples section. In another embodiment, biological activity can be assessed functionally, e.g., via measurement of expression of fibrosis associated genes and/or effect on a target cell such as fibroblasts, a fibrotic cell, a cancer cell, keratinocytes, melanocytes, adipocytes, neurons, etc. A representative method involving quantifying the amount of protein secreted from target cells such as normal or scar derived fibroblasts and cancer cells is provided in the Examples.

In one embodiment, the biologically active fragments comprise at least 100, 300, 500, 600, 800, 1000, 1200 or more contiguous amino acids of the Clostridial toxin. Fragments of the Clostridial toxins described herein can be generated by methods known to those skilled in the art (e.g., recombinant biotechnological techniques). Fragment polypeptides may be detected using art-known methods (e.g., immunogenic techniques such as immunoblotting or ELISA or protein staining techniques such as silver staining).

In certain embodiments, the fragment polypeptides may be secreted when expressed in a suitable host, e.g., baculovirus, E. coli, yeast, insect cell, or mammalian cells. As used herein, “secreted” means that the expressed polypeptide is secreted from the host cell into the culture medium at a level that is detectable using a conventional technique, e.g., ELISA assay. Methods for synthesizing proteins in secretory forms are known in the art. In one embodiment, the disclosure relates to Clostridial toxin fragments comprising the carboxy terminal regions of the heavy chains (H_(C) regions) comprising a Clostridial toxin binding domain, which upon binding to a receptor complex located at the surface of a target cell and modulates gene expression and/or metabolic activity of the target cell. The H_(C) regions from the heavy chains of Clostridial toxins are approximately 400-440 amino acids in length and comprise a binding domain (Table 1). Thus, aspects of this embodiment can include Clostridial toxin H_(C) regions comprising a binding domain having a length of, e.g., at least 20, 30, 40, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, or 425 amino acids. Other aspects of this embodiment can include Clostridial toxin H_(C) regions comprising a binding domain having a length of, e.g., at most 20, 30, 40, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, or 425 amino acids.

In one embodiment, the disclosure relates to the following binding domain fragments: amino acids N872-L1296 of BoNT/A (e.g., UNIPROT #P0DPI1); amino acids E859-E1291 of BoNT/B (e.g., UNIPROT #P10844); amino acids N867-E1291 of BoNT/C₁ (e.g., UNIPROT #P18640); amino acids S863-E1276 of BoNT/D (e.g., UNIPROT #P19321); amino acids R846-K1252 of BoNT/E (e.g., UNIPROT #Q00496 or GENBANK #CAA44558); amino acids K865-E1274 of BoNT/F (e.g., UNIPROT #P30996); amino acids N864-E1297 of BoNT/G (e.g., UNIPROT #Q60393); amino acids Y844-L1288 of BoNT/H (e.g., GENBANK #KG015617); amino acids I880-D1315 of TeNT (e.g., UNIPROT #P04958); amino acids I858-E1268 of BaNT (e.g., UNIPROT #Q45851); or amino acids K848-K1251 of BuNT (e.g., UNIPROT #P30995).

In another embodiment, the disclosure relates to a polypeptide comprising an amino acid sequence substantially identical to an amino acid sequence of the full-length botulinum toxin which is devoid of toxicity. In one embodiment, the polypeptide comprises a sequence of amino acids having at least 90% sequence identity to the full-length of a botulinum toxin which is devoid of toxicity.

In another embodiment, the polypeptide comprises an amino acid sequence substantially identical to an amino acid sequence of the carboxyl or C-terminal segment of the heavy chain of botulinum toxin (H_(C)). In one embodiment, the polypeptide comprises an amino acid sequence having at least 90% sequence identity to the C-terminal segment of the heavy chain of a botulinum toxin In one embodiment, the polypeptide comprises an amino acid sequence substantially identical to an amino acid sequence of the binding domain of the botulinum toxin. In one embodiment, the polypeptide comprises an amino acid sequence having at least 90% sequence identity to a binding domain of a botulinum toxin. In one embodiment, the polypeptide comprises an amino acid sequence substantially identical to an amino acid sequence of the N-terminal half of a binding domain of a botulinum toxin. In one embodiment, the polypeptide comprises an amino acid sequence having at least 90% sequence identity to the N-terminal half of a binding domain of a botulinum toxin.

In some embodiments, the botulinum toxin is selected from the group consisting of Botulinum toxin serotype A (BoNT/A), Botulinum toxin serotype B (BoNT/B), Botulinum toxin serotype C₁ (BoNT/C₁), Botulinum toxin serotype D (BoNT/D), Botulinum toxin serotype E (BoNT/E), Botulinum toxin serotype F (BoNT/F), Botulinum toxin serotype G (BoNT/G), Botulinum toxin serotype H (BoNT/H), Botulinum toxin serotype X (BoNT/X), and mosaic Botulinum toxins and/or variants thereof. Examples of mosaic toxins include BoNT/DC, BoNT/CD, and BoNT/FA. In one embodiment, the botulinum toxin is not Botulinum toxin serotype A (BoNT/A).

In another embodiment, the disclosure relates to Clostridial toxins fragments having an average molecular weight in the range of, e.g., about 1 kDa to about 160 kDa, about 5 kDa to about 160 kDa, about 20 kDa to about 150 kDa, about 40 kDa to about 120 kDa, about 50 kDa to about 80 kDa, about 5 kDa to about 15 kDa, about 10 kDa to about 20 kDa, about 20 kDa to about 30 kDa, about 30 kDa to about 40 kDa, about 40 kDa to about 50 kDa, about 50 kDa to about 60 kDa, about 60 kDa to about 70 kDa, about 70 kDa to about 80 kDa, about 80 kDa to about 90 kDa, about 90 kDa to about 100 kDa, about 100 kDa to about 110 kDa, about 110 kDa to about 120 kDa, about 120 kDa to about 130 kDa, about 130 kDa to about 140 kDa, about 140 kDa to about 150 kDa, or more, including all values in between, e.g., about 1 kDa, 2 kDa, 3 kDa, 4 kDa, 5 kDa, 6 kDa, 7 kDa, 8 kDa, 9 kDa, 10 kDa, 11 kDa, 12 kDa, 13 kDa, 14 kDa, 15 kDa, 16 kDa, 17 kDa, 18 kDa, 19 kDa, 20 kDa, 21 kDa, 22 kDa, 23 kDa, 24 kDa, 25 kDa, 26 kDa, 27 kDa, 28 kDa, 29 kDa, 30 kDa, 31 kDa, 32 kDa, 33 kDa, 34 kDa, 35 kDa, 36 kDa, 37 kDa, 38 kDa, 39 kDa, 40 kDa, 41 kDa, 42 kDa, 43 kDa, 44 kDa, 45 kDa, 46 kDa, 47 kDa, 48 kDa, 49 kDa, 50 kDa, 51 kDa, 52 kDa, 53 kDa, 54 kDa, 55 kDa, 56 kDa, 57 kDa, 58 kDa, 59 kDa, 60 kDa, 61 kDa, 62 kDa, 63 kDa, 64 kDa, 65 kDa, 66 kDa, 67 kDa, 68 kDa, 69 kDa, 70 kDa, 71 kDa, 72 kDa, 173 kDa, 74 kDa, 75 kDa, 76 kDa, 77 kDa, 78 kDa, 79 kDa, 80 kDa, 81 kDa, 82 kDa, 83 kDa, 84 kDa, 85 kDa, 86 kDa, 87 kDa, 88 kDa, 89 kDa, 90 kDa, 91 kDa, 92 kDa, 93 kDa, 94 kDa, 95 kDa, 96 kDa, 97 kDa, 98 kDa, 99 kDa, 100 kDa, 101 kDa, 102 kDa, 103 kDa, 104 kDa, 105 kDa, 106 kDa, 107 kDa, 108 kDa, 109 kDa, 110 kDa, 111 kDa, 112 kDa, 113 kDa, 114 kDa, 115 kDa, 116 kDa, 117 kDa, 118 kDa, 119 kDa, 120 kDa, 121 kDa, 122 kDa, 123 kDa, 124 kDa, 125 kDa, 126 kDa, 127 kDa, 128 kDa, 129 kDa, 130 kDa, 131 kDa, 132 kDa, 133 kDa, 134 kDa, 135 kDa, 136 kDa, 137 kDa, 138 kDa, 139 kDa, 140 kDa, 141 kDa, 142 kDa, 143 kDa, 144 kDa, 145 kDa, 146 kDa, 147 kDa, 148 kDa, 149 kDa, 150 kDa, or more, e.g., to about 155 kDa, about 160 kDa, about 165 kDa, about 170 kDa, about 175 kDa, about 180 kDa, about 190 kDa, about 200 kDa, about 225 kDa, about 250 kDa, about 275 kDa or more. In one embodiment, the disclosure relates to an N-terminal sub-domain (H_(CN)) of the binding domain of a Clostridial toxin having molecular weight in the range of, e.g., about 1 kDa to about 50 kDa, about 5 kDa to about 35 kDa, about 10 kDa to about 30 kDa, about 15 kDa to about 25 kDa, about 5 kDa to about 15 kDa, about 5 kDa to about 10 kDa, about 10 kDa to about 20 kDa, about 10 kDa to about 15 kDa, about 20 kDa to about 30 kDa, about 20 kDa to about 25 kDa, or more than 50 kDa, including all values in between, e.g., about 1 kDa, 2 kDa, 3 kDa, 4 kDa, 5 kDa, 6 kDa, 7 kDa, 8 kDa, 9 kDa, 10 kDa, 11 kDa, 12 kDa, 13 kDa, 14 kDa, 15 kDa, 16 kDa, 17 kDa, 18 kDa, 19 kDa, 20 kDa, 21 kDa, 22 kDa, 23 kDa, 24 kDa, 25 kDa, 26 kDa, 27 kDa, 28 kDa, 29 kDa, 30 kDa, 31 kDa, 32 kDa, 33 kDa, 34 kDa, 35 kDa, 36 kDa, 37 kDa, 38 kDa, 39 kDa, 40 kDa, 41 kDa, 42 kDa, 43 kDa, 44 kDa, 45 kDa, 46 kDa, 47 kDa, 48 kDa, 49 kDa, 50 kDa, or more. In one embodiment, the disclosure relates to a polypeptide comprising an amino acid sequence substantially identical to the amino or N-terminal half of the binding domain of the botulinum toxin (H_(CN)). In another embodiment, the disclosure relates to a polypeptide comprising an amino acid sequence substantially identical to an amino-terminal (N-terminal) half of the binding domain of a Clostridial toxin comprising the first 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250 or a greater number of contiguous amino acids from the N-terminal half of the binding domain of Clostridial toxins. In such embodiments, the polypeptide may have a molecular weight in the range of, e.g., about 1 kDa to about 25 kDa, about 5 kDa to about 20 kDa, about 6 kDa to about 15 kDa, about 8 kDa to about 15 kDa, about 8 kDa to about 14 kDa, about 6 kDa to about 25 kDa, about 8 kDa to about 20 kDa, about 10 kDa to about 15 kDa, including all values in between, e.g., about 1 kDa, 2 kDa, 3 kDa, 4 kDa, 5 kDa, 5.5 kDa, 6 kDa, 6.5 kDa, 7 kDa, 7.5 kDa, 8 kDa, 8.5 kDa, 9 kDa, 9.5 kDa, 10 kDa, 10.5 kDa, 11 kDa, 11.5 kDa, 12 kDa, 12.5 kDa, 13 kDa, 13.5 kDa, 14 kDa, 14.5 kDa, 15 kDa, 15.5 kDa, 16 kDa, 16.5 kDa, 17 kDa, 17.5 kDa, 18 kDa, 19 kDa, 20 kDa, or more.

In a related embodiment, the disclosure relates to a polypeptide comprising an amino acid sequence substantially identical to the C-terminal sub-domain (H_(CC)) of the binding domain of a Clostridial toxin having molecular weight in the range of, e.g., about 1 kDa to about 50 kDa, about 5 kDa to about 35 kDa, about 10 kDa to about 30 kDa, about 15 kDa to about 25 kDa, about 5 kDa to about 15 kDa, about 5 kDa to about 10 kDa, about 10 kDa to about 20 kDa, about 10 kDa to about 15 kDa, about 20 kDa to about 30 kDa, about 20 kDa to about 25 kDa, or more than 50 kDa, including all values in between, e.g., about 1 kDa, 2 kDa, 3 kDa, 4 kDa, 5 kDa, 6 kDa, 7 kDa, 8 kDa, 9 kDa, 10 kDa, 11 kDa, 12 kDa, 13 kDa, 14 kDa, 15 kDa, 16 kDa, 17 kDa, 18 kDa, 19 kDa, 20 kDa, 21 kDa, 22 kDa, 23 kDa, 24 kDa, 25 kDa, 26 kDa, 27 kDa, 28 kDa, 29 kDa, 30 kDa, 31 kDa, 32 kDa, 33 kDa, 34 kDa, 35 kDa, 36 kDa, 37 kDa, 38 kDa, 39 kDa, 40 kDa, 41 kDa, 42 kDa, 43 kDa, 44 kDa, 45 kDa, 46 kDa, 47 kDa, 48 kDa, 49 kDa, 50 kDa, or more.

In another embodiment, the present disclosure provides for Clostridial toxins fragments having the aforementioned molecular weights, e.g., between about 1 kDa to about 150 kDa, particularly between about 5 kDa to about 90 kDa, especially between about 10 kDa to about 70 kDa, as determined by reducing gel electrophoresis.

In other embodiment, the molecular weight of the Clostridial toxins, including fragments thereof, are theoretically computed using art-known bioinformatics tools (e.g., PROTPARAM or COMPUTE p1/MW). For instance, based on PROTPARAM, the full-length BoNT/A (UNIPROT #P0DPI1) has a theoretical molecular weight of about 149.3 kDa, while the H_(C) domain comprising amino acids 449 to 1296 (848 amino acids) has a theoretical MW of about 98.2 kDa. In a particular embodiment, an H_(CN) domain of BoNT/A (UNIPROT #P0DPI1) spanning amino acids 873 to 1092 has a theoretical molecular weight of about 26.1 kDa based on PROTPARAM. Still in a further embodiment, the proximal N-terminal fragment of an H_(CN) domain of BoNT/A (UNIPROT #P0DPI1) spanning amino acids 873 to 980 has a theoretical molecular weight of about 12.65 kDa.

Representative amino acid sequences of the BoNT H_(C) domains and H_(CN) domains derived from various serotypes of BoNT, which were used in the sequence alignment analysis for the identification of identical and consensus sequences, are identified herein as SEQ ID NOs: 3-9.

Modifications

In some cases, a Clostridial toxin (or a domain or a sub-domain thereof) comprises one or more modifications. For example, the Clostridial toxin, or fragments thereof, can be cyclized. As another example, the Clostridial toxin, or fragments thereof, can have one or more amino acid modifications, e.g., inclusion of one or more D-amino acids. Modifications of interest that do not alter primary sequence include chemical derivatization of polypeptides, e.g., acetylation or carboxylation. Also included are modifications of glycosylation, e.g. those made by modifying the glycosylation patterns of a polypeptide during its synthesis and processing or in further processing steps; e.g., by exposing the polypeptide to enzymes which affect glycosylation, such as mammalian glycosylating or deglycosylating enzymes. Also embraced are polypeptides that have phosphorylated amino acid residues, e.g., phosphotyrosine, phosphoserine, or phosphothreonine.

Also provided are Clostridial toxins, or fragments thereof, that have been modified using ordinary molecular biological techniques and/or synthetic chemistry so as to improve their resistance to proteolytic degradation or to optimize solubility properties or to render them more suitable as a therapeutic agent. Analogs of such polypeptides include those containing residues other than naturally occurring L-amino acids, e.g., D-amino acids or non-naturally occurring synthetic amino acids.

A toxin may be joined to a wide variety of other oligopeptides or proteins for a variety of purposes. By providing for expression of the subject polypeptides, various post-translational modifications may be achieved. For example, by employing the appropriate coding sequences, one may provide farnesylation or prenylation. For example, the toxin can be bound to a lipid group at a terminus, so as to be able to be bound to a lipid membrane, such as a liposome.

Other suitable modifications on the Clostridial toxin or fragments thereof include, e.g., (1) end-cappings of the terminal of the polypeptides, such as amidation of the C-terminus and/or acetylation or deamination of the N-terminus; (2) introducing peptidomimetic elements in the structure; and (3) cyclization, in which the cyclization of the polypeptide can occur through natural amino acids or non-naturally-occurring building blocks.

A modified Clostridial toxin or a fragment thereof can be a peptoid (N-substituted oligoglycines), e.g., in which an amino acid side chain is connected to the nitrogen of the polypeptide backbone, instead of the α-carbon. See, e.g., Zuckermann et al., J. Am. Chem. Soc. 114, 10646, 1992).

A subject toxin can include naturally-occurring and non-naturally occurring amino acids. A Clostridial toxin or a fragment thereof can comprise D-amino acids, a combination of D- and L-amino acids, and various “designer” amino acids (e.g., β-methyl amino acids, Cα-methyl amino acids, and Nα-methyl amino acids, etc.) to convey special properties to polypeptides.

Fusion Proteins and Linked Proteins

It is understood that a modified Clostridial toxin or a fragment thereof disclosed in the present specification can optionally include one or more additional components. As a non-limiting example of an optional component, a modified Clostridial toxin or a fragment thereof can further comprise a flexible region comprising a flexible spacer. Non-limiting examples of a flexible spacer include, e.g., a G-spacer GGGGS (SEQ ID NO: 22) or an A-spacer EAAAK (SEQ ID NO: 23). A flexible region comprising flexible spacers can be used to adjust the length of a polypeptide region in order to optimize a characteristic, attribute or property of a polypeptide. Such a flexible region is operably-linked in-frame to the modified Clostridial toxin or a fragment thereof as a fusion protein. As a non-limiting example, a polypeptide region comprising one or more flexible spacers in tandem can be used to better expose a protease cleavage site thereby facilitating cleavage of that site by a protease. As another non-limiting example, a polypeptide region comprising one or more flexible spacers in tandem can be used to better present a ligand domain, thereby facilitating the binding of that ligand domain to its binding domain on a receptor.

Thus, in an embodiment, a modified Clostridial toxin or a fragment thereof disclosed in the present specification can further comprise a flexible region comprising a flexible spacer. In another embodiment, a modified Clostridial toxin or a fragment thereof disclosed in the present specification can further comprise flexible region comprising a plurality of flexible spacers in tandem. In aspects of this embodiment, a flexible region can comprise in tandem, e.g., at least 1 G-spacer, at least 2 G-spacers, at least 3 G-spacers, at least 4 G-spacers or at least 5 G-spacers. In other aspects of this embodiment, a flexible region can comprise in tandem, e.g., at most 1 G-spacer, at most 2 G-spacers, at most 3 G-spacers, at most 4 G-spacers or at most 5 G-spacers. In still other aspects of this embodiment, a flexible region can comprise in tandem, e.g., at least 1 A-spacer, at least 2 A-spacers, at least 3 A-spacers, at least 4 A-spacers or at least 5 A-spacers. In still other aspects of this embodiment, a flexible region can comprise in tandem, e.g., at most 1 A-spacer, at most 2 A-spacers, at most 3 A-spacers, at most 4 A-spacers or at most 5 A-spacers. In another aspect of this embodiment, a modified Clostridial toxin or a fragment thereof can comprise a flexible region comprising one or more copies of the same flexible spacers, one or more copies of different flexible-spacer regions, or any combination thereof.

Properties of the Clostridial Toxins Lack of Toxicity Profile

In one embodiment, the present disclosure contemplates compositions and methods directed to Clostridial toxins (e.g., BoNT/A) or fragments thereof of modified toxicity, including reduced toxicity or devoid of toxicity. As is known in the art, toxic activity of Clostridial toxins is particularly contained in the light chain, which is a zinc (Zn²⁺) endopeptidase that selectively cleaves soluble NSF attachment protein receptor (“SNARE”) proteins. SNARE proteins are important for recognition and docking of neurotransmitter-containing vesicles with the cytoplasmic surface of the plasma membrane, and fusion of the vesicles with the plasma membrane. TeNT, BoNT/B BoNT/D, BoNT/F, and BoNT/G cause degradation of synaptobrevin (also called vesicle-associated membrane protein (VAMP)), a synaptosomal membrane protein. Most of the cytosolic domain of VAMP extending from the surface of the synaptic vesicle is removed as a result of any one of these cleavage events. BoNT/A and BoNT/E selectively cleave the plasma membrane-associated protein SNAP-25, which is predominantly bound to and present on the cytosolic surface of the plasma membrane. BoNT/C cleaves syntaxin, an integral protein having most of its mass exposed to the cytosol. Syntaxin interacts with the calcium channels at presynaptic terminal active zones. In some embodiments, the toxicity of the Clostridial toxin or a fragment thereof is assayed in terms of induction of neuromuscular paralysis (which is an indication of the toxin molecules' ability to enter the cell and thence to inhibit neurotransmitter release. In some embodiments, the toxicity of Clostridial toxin or a fragment thereof is assayed in terms of LD₅₀ values, which is the amount of toxin that induces death in 50% (one half) of a group of test animals, e.g., mice, upon intraperitoneal injection of the toxin construct. See, U.S. Pat. Nos. 7,749,514 and 9,284,545.

In aspects of this embodiment, the Clostridial toxin or a fragment thereof is, e.g., about 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or >99% as toxic as a naturally-occurring Clostridial toxin. In aspects of this embodiment, the modified Clostridial toxin or a fragment thereof is, e.g., at most 10% as toxic as a naturally-occurring Clostridial toxin, at most 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or <95% as toxic as a naturally-occurring Clostridial toxin. Preferably, the Clostridial toxin fragments or variants of the disclosure are devoid of toxicity, e.g., when applied in a conventional manner to a subject.

In one embodiment, the Clostridial toxins of modified toxicity or fragments thereof disclosed herein finds applications including, but not limited to: (a) research (i.e., for example, into the mechanism of action of BoNT/A, including its binding, translocation, and pharmacokinetics, and for its use to develop and test an antidote); (c) assessing risks and diagnostics for indoor release; (d) for examining pharmacokinetics in mammals, including primates; (d) vaccine development; (e) antibody development (for therapy and diagnostics); and (f) clinical therapeutic applications.

Toxicities of Clostridial toxins can be assessed using routine methods. As is known in the art, a multi-step mechanism is involved in the cell intoxication by BoNTs (Chaddock et al., Trends Biochem. Sci. 27, 552-558, 2002). The neurotoxin binds to the pre-synaptic nerve endings of neurons through a heavy chain (H) and enters by receptor-mediated endocytosis (Schiand et al., Physio. Rev. 80, 717-755, 2000). The low pH of endosome is believed to induce channel formation by the H_(CN), which allows translocation of the LC into the cytosol (Li et al., Biochemistry 39, 6466-6474, 2000). It is believed that LC works as a zinc endopeptidase to cleave specifically one of the three different SNARE proteins essential for synaptic vesicle fusion (Montecucco et al., Trends Biochem. Sci. 18, 324-327, 1993; Li et al., Toxin Rev. 18, 95-112, 1999). In one embodiment, BoNT/A and BoNT/E, independently, cleave synaptosomal-associated protein 25 (SNAP-25), a component of the trans-SNARE complex, which is proposed to account for the specificity of membrane fusion and to directly execute fusion by forming a tight complex that brings the synaptic vesicle and plasma membranes together. In another embodiment, TeNT and/or BoNT/B, /D, /F and /G cleave cellubrevin, a protein involved in the docking and/or fusion of synaptic vesicles with the presynaptic membrane. In one embodiment, BoNT/C₁ cleaves syntaxin and SNAP-25. Once a SNARE protein is cleaved, the release of a neurotransmitter (i.e., for example, acetylcholine) is prevented, ultimately leading to the flaccid muscle paralysis (Montecucco et al., Q. Rev. Biophys, 28:423-472 (1995). The botulinum neurotoxin active site is believed to comprise of a HEXXH+E zinc-binding motif (Li et al., Biochemistry 39, 2399-2405, 2000). The general conformation and active site residues appear conserved in all of the Clostridial neurotoxins (Agarwal et al., Biochemistry 44, 8291-8302, 2005). For example, the amino acid residues in BoNT/A active site comprise H223-E224-L225-I226-H227+E262 (“H223-E224-L225-I226-H227” disclosed as SEQ ID NO: 24), which are conserved in most, if not all, BoNT subtypes.

Accordingly, the present disclosure relates to a non-toxic form of Clostridial toxin that is devoid of endopeptidase activity or translocation activity or both endopeptidase activity and translocation activity. In one embodiment, provided herein are Clostridial toxin variants lacking endopeptidase activity. Such variants may comprise, for example, mutations or deletions of one or more amino acid residues making up the active site, which confers endopeptidase activity. In another embodiment, the Clostridial toxin variants lacking endopeptidase activity may comprise deletion of a substantial portion, e.g., deletion of about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or >99%, of the amino acids making up the light chain (LC) domain. In another embodiment, the Clostridial toxin variant is devoid of both endopeptidase activity and translocation activity. In this embodiment, the Clostridial toxin variant may comprises deletion of a substantial portion of (a) the amino acids making up the light chain (LC) domain and (b) the amino acids making up the translocation domain.

In the context of BoNT/A, the disclosure contemplates fragments that do not cleave SNAP-25. Reagents and assays for measuring SNAP-25 cleavage activity are known in the art. See, e.g., Mizanur et al., PLoS One, 9(4), e95188, 2014.

The disclosure further relates to inactive Clostridial toxins, including fragments thereof. The term “inactive Clostridial toxin” means a Clostridial toxin that is not toxic to a cell. For example, an inactive Clostridial toxin has minimal or no ability to interfere with the release of neurotransmitters from a cell or nerve endings. In some embodiments, the inactive Clostridial toxin has less than about 50%, e.g., about 40%, about 25%, about 10%, about 5%, or a lesser %, e.g., about 2%, of the neurotoxic effect (e.g., ability to inhibit release of neurotransmitter) of an identical Clostridial toxin that is active. For example, an inactive botulinum toxin (iBoNT) has less than about 50%, e.g., about 40%, about 25%, about 10%, about 5%, or a lesser %, e.g., about 2%, of the neurotoxic effect of an identical BoNT that is active (e.g., full-length BoNT). Full-length inactive botulinum toxins are disclosed in, e.g., U.S. Pat. Nos. 6,051,239 and 7,172,764. In some embodiments, the inactive Clostridial toxin comprises a heavy chain that is modified (e.g., glycosylated) as to reduce antigenicity. In some embodiments, inactive Clostridial toxin is a single chain polypeptide. Preferably, the inactive Clostridial toxin of the disclosure comprises a fragment of full length Clostridial toxin that is devoid of the light chain (LC) domain and/or the translocation domain, wherein the fragment Clostridial toxin is further optionally glycosylated so as to reduce antigenicity.

The term “reduced antigenicity,” as used herein, means the ability of the inactive Clostridial toxin to induce the production of antibody in a mammal is less than the antigenic effect of a full-length Clostridial toxin, e.g., less than about 100%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, e.g., about 40%, about 25%, about 10%, about 5%, or a lesser %, e.g., about 2%, of the antigenic effect of a full-length Clostridial toxin. For example, molecules which are glycosylated may have reduced antigenicity because they have minimal or no ability to induce an immune response for the production of antibody in a mammal. Also, epitope regions on a molecule are responsible for the induction of antibodies in a mammal. Thus, molecules with epitope regions mutated or deleted may have reduced antigenicity because these regions are no longer present on the molecule to stimulate antibody production. See, U.S. Pat. No. 7,172,764. For example, an iBoNT comprising a mutated or deleted epitope region within its heavy chain at the carboxy terminal (H_(C)) can have a reduced antigenicity compared to full-length Clostridial toxin. In some embodiments, the administration of a glycosylated BoNT into a mammal induces less production of antibody as compared to an administration of an identical BoNT which is not glycosylated, by about 2-fold, preferably 4-fold, more preferably 8-fold, or more. The antigenicity of the Clostridial toxins of the instant disclosure can be determined using methods and tools known in the art, e.g., Atassi et al., Protein J 23(1):39-52, 2004.

Embodiments disclosed herein further relate to methods for assaying for the toxicity of Clostridial toxins, including fragments and variants thereof, comprising, e.g., biochemical assays, in vitro cell-based assays, in vivo pharmacological assays, and the like. In one embodiment, the Clostridial toxin fragment or variant lacks endopeptidase activity in a standard endopeptidase assay (see, U.S. Pat. Nos. 8,618,261; 8,067,231; 8,124,357; 7,645,570 for variations in the in vitro assay). In another embodiment, the Clostridial toxin fragment or variant is non-lethal.

Modulation of Cell Signaling

In a related embodiment, provided herein are Clostridial toxins, sub-domains or fragments thereof or variants thereof, which modulate the expression of one or more fibrosis associated genes, including but not limited to for example FGFR1, MMP1, MMP3, TIMP1, FGF7, TP63, SOD2, UBD, HAS2, HAS3, ADAMTS1, IGF-1, IL-6, IL-32, CCL2, BDKRB1, S100A4 and ACTA2.

Changes in expression of these fibrosis associated genes affect structural and functional characteristics of fibrosis associated cells, tissues and/or organs, including the extracellular matrix structure, resulting in changes in fibroblast proliferation, fibroblast to myofibroblasts transition, fibroblast contractility, production and/or secretion of Extra Cellular Matrix (ECM) components, such as for example collagens, fibrin, fibronectin, elastin, proteoglycans, glycosaminoglycans, and matrix cellular proteins production/secretion. For example, as shown in FIG. 1, treatment of normal fibroblast with the polypeptide provided according to the present method increased expression of genes involved in ECM repair and remodeling, such as for example MMP1 and MMP3, and reduced expression of genes associated with fibroblast contractility and myofibroblast formation, such as for example S100A4 and ACTA2. Since fibrosis and formation of scars are associated with fibroblast contractility, myofibroblast formation, the results obtained show that polypeptides provided according to the present method have anti-scarring and anti-fibrotic effects. By “modulate,” it is meant that the Clostridial toxin or a fragment or variant thereof, when contacted with a target cell, e.g., fibroblast, keratinocyte, melanocyte, sebocyte, immune cells, osteoblasts, chondrocytes, myocytes, adipocytes, glial cells or neuron, effectuates a change of at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 1-fold, at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 30-fold, at least 50-fold, or more, in the expression of one or more of the aforementioned genes compared to a control (e.g., BSA treatment). Methods of measuring gene expression are known in the art, e.g., microarray analysis or quantitative PCR assay. Representative methods are illustrated in the Examples section.

Particularly, provided herein are Clostridial toxins, subdomains or fragments or variants thereof, which, when contacted with a target cell, e.g., normal fibroblast or scar derived fibroblast, fibrotic cell, cancer cell modulate the expression of one or more genes selected from FGFR1, MMP1, MMP3, TIMP1, FGF7, TP63, S100A4 and ACTA2. Especially, the Clostridial toxins or fragments or variants thereof modulate (e.g. increase) the expression of each gene in the aforementioned six-gene signature by at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 1-fold, at least 1.5-fold, at least 2-fold, at least 2.5-fold, or more. Changes in expression of these extracellular matrix associated genes affect production or secretion of proteins involved in creating, maintaining and repairing the dermis Extra Cellular Matrix (ECM). Since formation of scars and fibrosis are associated with excess production of some ECM components, the results obtained show that polypeptides provided according to the present method have anti-scarring and anti-fibrotic effects.

Additionally, the disclosure relates to Clostridial toxins or fragments or variants thereof which modulates the expression or secretion of fibronectin. “Fibronectin,” as used herein, refers to a high-molecular weight (˜440 kDa) glycoprotein of the extracellular matrix that binds to component proteins in the ECM, e.g., collagen, fibrin, and heparan sulfate proteoglycans. In one embodiment, the Clostridial toxins or fragments or variants thereof, when contacted with a cell, e.g., fibroblast, modulates the expression or secretion of fibronectin by at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 1-fold, at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 5-fold, or more. Changes in fibronectin expression/secretion affects the structure and function of the dermis, including the extracellular matrix structure, resulting in anti-fibrotic effects.

Effect on Target Cells

The disclosure further relates to Clostridial toxins or fragments or variants thereof which change one or more features of the target cells, e.g., cells to which they bind. In one embodiment, the feature is a functional attribute such as fibroblast contractility, migration, differentiation, secretion (e.g., ECM components) and adhesion (to each other and to the matrix). In yet another embodiment, the feature is modulated expression of a gene such as a gene involved in ECM repair. In a particular embodiment, the feature is attenuated production or secretion of a protein in the presence of a mediator, e.g., attenuation of fibronectin and collagen secretion from scar derived fibroblasts. As shown in the Examples, when contacted with the target cells, e.g., fibroblast cells, the Clostridial toxin fragments effectuate an appreciable change in specific protein secretion and/or gene expression. Additionally, treating hypoxia-induced fibrotic cancer cells with the Clostridial toxin fragments resulted in the same effects on protein secretion as known anti-fibrotic agents, e.g., FG-3019.

The cellular effects of the Clostridial toxins or fragments or variants thereof can be assayed using techniques that are described in detail in the Examples. A variety of target cells, e.g., fibroblasts, keratinocytes, adipocytes, osteoblasts, chondrocytes, myocytes, glial cells, neurons; cell-lines; and tissues including connective, epithelial, and endothelial tissues may be used to assay for the effect of the Clostridial toxins or fragments or variants thereof at the cellular level.

Polynucleotides

Aspects of the present disclosure provide, in part, polynucleotide molecules. As used herein, the term “polynucleotide molecule” is synonymous with “nucleic acid molecule” and means a polymeric form of nucleotides, such as, e.g., ribonucleotides and deoxyribonucleotides, of any length. It is envisioned that any and all polynucleotide molecules that can encode a modified Clostridial toxin disclosed in the present specification can be useful, including, without limitation naturally-occurring and non-naturally-occurring DNA molecules and naturally-occurring and non-naturally-occurring RNA molecules. Non-limiting examples of naturally-occurring and non-naturally-occurring DNA molecules include single-stranded DNA molecules, double-stranded DNA molecules, genomic DNA molecules, cDNA molecules, vector constructs, such as, e.g., plasmid constructs, phagemid constructs, bacteriophage constructs, retroviral constructs and artificial chromosome constructs. Non-limiting examples of naturally-occurring and non-naturally-occurring RNA molecules include single-stranded RNA, double stranded RNA and mRNA.

In one embodiment, the polynucleotide molecules encode one or more of the aforementioned Clostridial toxins, mutants or variants thereof, domains and/or sub-domains thereof, biologically-active or immunogenic fragments thereof, multimers thereof, chimeras and fusion constructs thereof, tagged constructs thereof, mimetics thereof, or other forms of engineered or synthetic derivatives thereof. Particularly, the polynucleotide molecule is a DNA molecule and especially, the polynucleotide is a cDNA molecule. Also included are polynucleotides which are complementary to the polynucleotides encoding one or more of the aforementioned Clostridial toxins, including, mutants, variants, or fragments thereof. Especially, the polynucleotide is a cDNA molecule encoding the cell-binding domain of BoNT/A, including, homologs thereof, e.g., cell-binding domains of BoNT/B, BoNT/C₁, BoNT/D, BoNT/E, BoNT/F, BoNT/G, BoNT/H, TeNT, BaNT or BuNT. Particularly preferably, the polynucleotide is a cDNA molecule encoding the heavy chain N-terminal sub-domain (H_(CN)) of cell-binding domain of BoNT/A or a mutant thereof, including, homologs thereof, e.g., H_(CN) sub-domains of BoNT/B, BoNT/C₁, BoNT/D, BoNT/E, BoNT/F, BoNT/G, BoNT/H, BoNT/X, eBoNT/J, TeNT, BaNT or BuNT.

The present disclosure also provides synthetic nucleic acids, e.g., non-natural nucleic acids, comprising nucleotide sequence encoding one or more of the aforementioned Clostridial toxins, including fragments thereof.

Included herein are nucleic acids encoding Clostridial toxin fragment sequences set forth in SEQ ID NOs: 1, 19, SEQ ID NOs: 3-18, or a variant thereof having 1, 2, 3, 4, 5, 7, 10, 15, 20, 25, 30 or more amino substitutions (preferably conserved or semi-conserved amino acid substitutions), or a homolog thereof, including the complementary strand thereto, or the RNA equivalent thereof, or a complementary RNA equivalent thereof.

Also, included herein are nucleic acids encoding Clostridial toxin fragment sequences of SEQ ID NOs: 1, 19, SEQ ID NOs: 3-18, or a variant thereof having 1, 2, 3, 4, 5, 4-10, 5-10, or 5 to 15 amino acid substitutions (preferably conserved or semi-conserved amino acid substitutions), or a homolog thereof, including the complementary strand thereto, or the RNA equivalent thereof, or a complementary RNA equivalent thereof. In some embodiments, the nucleic acids of the disclosure encode fragments of BoNT/A mutants, comprising, consisting of, or consisting of the sequence set forth in SEQ ID NO: 25, SEQ ID NO: 26, or SEQ ID NO: 27.

The disclosure further relates to nucleic acids homologs of Clostridial toxins fragments, e.g., a fragment which encodes the H_(C) domain, particularly the H_(CN) domain and especially the N-terminal half of the H_(CN) domain of a Clostridial toxin selected from the group consisting of BoNT/A, BoNT/B, BoNT/C₁, BoNT/D, BoNT/E, BoNT/F, BoNT/G, BoNT/H, BoNT/DC, BoNT/X, eBoNT/J, TeNT, BaNT, BuNT, including the aforementioned subtypes thereof. Sequences having substantial homology include nucleic acid sequences having at least 50%, particularly at least 65%, and especially at least 80% identity or greater % identity with the sequences as shown in SEQ ID NO: 2 or a nucleic acid encoding SEQ ID NOs: 1,19, or anyone of SEQ ID NOs: 3-18. Sequence identity can be calculated according to methods known in the art, e.g., using BLAST v2.1. See also, Altschul et al., J. Mol. Biol. 215:403-410, 1990; Gish et al., Nature Genet. 3:266-272, 1993; Madden et al., Meth. Enzymol. 266:131-141, 1996; Altschul et al., Nucleic Acids Res. 25:3389-3402, 1997; Zhang et al., Genome Res. 7:649-656, 1997.

Embodiments disclosed herein further relate to methods of making the above-disclosed polynucleotides. Well-established molecular biology techniques that may be necessary to make a polynucleotide molecule encoding a modified Clostridial toxin disclosed in the present specification including, but not limited to, procedures involving polymerase chain reaction (PCR) amplification, restriction enzyme reactions, agarose gel electrophoresis, nucleic acid ligation, bacterial transformation, nucleic acid purification, nucleic acid sequencing and recombination-based techniques are routine procedures well within the scope of one skilled in the art and from the teaching herein. Non-limiting examples of specific protocols necessary to make a polynucleotide molecule encoding a modified Clostridial toxin are described in e.g., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Frederick M. Ausubel et al., eds. John Wiley & Sons, 2004). Additionally, a variety of commercially available products useful for making a polynucleotide molecule encoding a modified Clostridial toxin are widely available. These protocols are routine procedures well within the scope of one skilled in the art and from the teaching herein.

Another aspect of the present disclosure provides a method of producing a Clostridial toxin or fragments or variants thereof comprising, e.g., the steps of introducing an expression construct comprising a polynucleotide molecule encoding the Clostridial toxin or a fragment or variant thereof into a cell and expressing the expression construct in the cell.

The methods disclosed in the present specification include, in part, all Clostridial toxins or fragments or variants thereof disclosed in the present specification. Thus, aspects of this embodiment include producing, without limitation, naturally occurring Clostridial toxins, naturally occurring Clostridial toxins variants, such as, e.g., Clostridial toxins isoforms and Clostridial toxins subtypes, non-naturally occurring Clostridial toxins variants, such as, e.g., conservative or semi-conservative Clostridial toxins variants, non-conservative Clostridial toxins variants, chimeric or fusion constructs comprising one or more of the aforementioned toxins, Clostridial toxins fragments, e.g., biologically active fragments or immunogenic fragments, comprising at least one domain (for example the binding domain of the heavy chain) or a sub-domain of Clostridial toxin (for example the N-terminal half of the binding domain), chimeric or fusion constructs comprising such Clostridial toxins or fragments or variants thereof, tagged constructs, engineered constructs, synthetic constructs, or any combination thereof.

The methods disclosed in the present specification include, in part, a polynucleotide molecule. Particularly, the polynucleotide molecule encodes any Clostridial toxin or a fragment or variant thereof disclosed herein, including, a fusion protein comprising the Clostridial toxin or a fragment or variant thereof. It is envisioned that any and all polynucleotide molecules disclosed in the present specification can be used. Thus, aspects of this embodiment include, without limitation, naturally-occurring and non-naturally-occurring DNA molecules include single-stranded DNA molecules, double-stranded DNA molecules, genomic DNA molecules, cDNA molecules, vector constructs, such as, e.g., plasmid constructs, phagemid constructs, bacteriophage constructs, retroviral constructs and artificial chromosome constructs. Non-limiting examples of naturally-occurring and non-naturally-occurring RNA molecules include single-stranded RNA, double stranded RNA and mRNA.

The methods disclosed in the present specification include, in part, an expression construct. An expression construct comprises a polynucleotide molecule disclosed in the present specification operably-linked to an expression vector useful for expressing the polynucleotide molecule in a cell or cell-free extract. A wide variety of expression vectors can be employed for expressing a polynucleotide molecule encoding a modified Clostridial toxin or a fragment or variant thereof, including, without limitation, a viral expression vector; a prokaryotic expression vector; eukaryotic expression vectors, such as, e.g., a yeast expression vector, an insect expression vector and a mammalian expression vector; and a cell-free extract expression vector. It is further understood that expression vectors useful to practice aspects of these methods may include those which express a modified Clostridial toxin or a fragment or variant thereof under control of a constitutive, tissue-specific, cell-specific or inducible promoter element, enhancer element or both. Non-limiting examples of expression vectors, along with well-established reagents and conditions for making and using an expression construct from such expression vectors are readily available from commercial vendors. The selection, making and use of an appropriate expression vector are routine procedures well within the scope of one skilled in the art and from the teachings herein.

Thus, aspects of this embodiment include, without limitation, a viral expression vector operably-linked to a polynucleotide molecule encoding a modified Clostridial toxin or a fragment or variant thereof a prokaryotic expression vector operably-linked to a polynucleotide molecule encoding a modified Clostridial toxin or a fragment or variant thereof a yeast expression vector operably-linked to a polynucleotide molecule encoding a modified Clostridial toxin or a fragment or variant thereof an insect expression vector operably-linked to a polynucleotide molecule encoding a modified Clostridial toxin or a fragment or variant thereof and a mammalian expression vector operably-linked to a polynucleotide molecule encoding a modified Clostridial toxin or a fragment or variant thereof. Other aspects of this embodiment include, without limitation, expression constructs suitable for expressing a modified Clostridial toxin or a fragment or variant thereof disclosed in the present specification using a cell-free extract comprising a cell-free extract expression vector operably linked to a polynucleotide molecule encoding a modified Clostridial toxin or a fragment or variant thereof.

The methods disclosed in the present specification include, in part, a cell. It is envisioned that any and all cells can be used. Thus, aspects of this embodiment include, without limitation, prokaryotic cells including, without limitation, strains of aerobic, microaerophilic, capnophilic, facultative, anaerobic, gram-negative and gram-positive bacterial cells such as those derived from, e.g., Escherichia coli, Bacillus subtilis, Bacillus licheniformis, Bacteroides fragilis, Clostridia perfringens, Clostridia difficile, Caulobacter crescentus, Lactococcus lactis, Methylobacterium extorquens, Neisseria meningirulls, Neisseria meningitidis, Pseudomonas fluorescens and Salmonella typhimurium; and eukaryotic cells including, without limitation, yeast strains, such as, e.g., those derived from Pichia pastoris, Pichia methanolica, Pichia angusta, Schizosaccharomyces pombe, Saccharomyces cerevisiae and Yarrowia lipolytica; insect cells and cell lines derived from insects, such as, e.g., those derived from Spodoptera frugiperda, Trichoplusia ni, Drosophila melanogaster and Manduca Sexta; stinging cells (specifically, cnidocytes, nematocytes, or ptychocytes) of an organism belonging to the phylum Cnidaria (for example, hydras, sea anemones, jellyfish or corals, e.g., Aiptasia sp.) and/or a genetically transformed organism from the phylum Cnidaria; see, U.S. Pat. No. 6,923,976) and mammalian cells and cell-lines derived from mammalian cells, such as, e.g., those derived from mouse, rat, hamster, porcine, bovine, equine, primate and human. Cell lines may be obtained from the American Type Culture Collection (2004); European Collection of Cell Cultures (2204); and the German Collection of Microorganisms and Cell Cultures (2004). Non-limiting examples of specific protocols for selecting, making and using an appropriate cell line are described in e.g., INSECT CELL CULTURE ENGINEERING (Mattheus F. A. Goosen et al. eds., Marcel Dekker, 1993); INSECT CELL CULTURES: FUNDAMENTAL AND APPLIED ASPECTS (J. M. Vlak et al. eds., Kluwer Academic Publishers, 1996); Maureen A. Harrison & Ian F. Rae, GENERAL TECHNIQUES OF CELL CULTURE (Cambridge University Press, 1997); CELL AND TISSUE CULTURE: LABORATORY PROCEDURES (Alan Doyle et al., eds., John Wiley and Sons, 1998); R. Ian Freshney, CULTURE OF ANIMAL CELLS: A MANUAL OF BASIC TECHNIQUE (Wiley-Liss, 4^(th) ed. 2000); ANIMAL CELL CULTURE: A PRACTICAL APPROACH (John R. W. Masters ed., Oxford University Press, 3^(rd) ed. 2000); MOLECULAR CLONING A LABORATORY MANUAL, supra, (2001); BASIC CELL CULTURE: A PRACTICAL APPROACH (John M. Davis, Oxford Press, 2^(nd) ed. 2002); and CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, supra, (2004). Wherein the cell is a Cnidarian cell, it can be transformed using routine techniques of electroporation or using double-stranded RNA. See, Wittlieb et al., PNAS USA, 103(16):6208-11, 2006; Pankow et al., PLoS One, 2(9):e782, 2007; Khalturin et al., PLoS Biol., 6(11):e278, 2008. These protocols are routine procedures within the scope of one skilled in the art and from the teaching herein.

The methods disclosed in the present specification include, in part, introducing a polynucleotide molecule into a cell. A polynucleotide molecule introduced into a cell can be transiently or stably maintained by that cell. Stably-maintained polynucleotide molecules may be extra-chromosomal and replicate autonomously, or they may be integrated into the chromosomal material of the cell and replicate non-autonomously. It is envisioned that any and all methods for introducing a polynucleotide molecule disclosed in the present specification into a cell can be used. Methods useful for introducing a nucleic acid molecule into a cell include, without limitation, chemical-mediated transfection such as, e.g., calcium phosphate-mediated, diethyl-aminoethyl (DEAE) dextran-mediated, lipid-mediated, polyethyleneimine (PEI)-mediated, polylysine-mediated and polybrene-mediated; physical-mediated transfection, such as, e.g., biolistic particle delivery, microinjection, protoplast fusion and electroporation; and viral-mediated transfection, such as, e.g., retroviral-mediated transfection, see, e.g., Introducing Cloned Genes into Cultured Mammalian Cells, pp. 16.1-16.62 (Sambrook & Russell, eds., Molecular Cloning A Laboratory Manual, Vol. 3, 3^(rd) ed. 2001). One skilled in the art understands that selection of a specific method to introduce an expression construct into a cell will depend, in part, on whether the cell will transiently contain an expression construct or whether the cell will stably contain an expression construct. These protocols are routine procedures within the scope of one skilled in the art and from the teaching herein.

In an aspect of this embodiment, a chemical-mediated method, termed transfection, is used to introduce a polynucleotide molecule encoding a modified Clostridial toxin or a fragment or variant thereof into a cell. In chemical-mediated methods of transfection the chemical reagent forms a complex with the nucleic acid that facilitates its uptake into the cells. Such chemical reagents include, without limitation, calcium phosphate-mediated, see, e.g., Martin Jordan & Florian Worm, Transfection of Adherent and Suspended Cells by Calcium Phosphate, 33(2) Methods 136-143 (2004); diethyl-aminoethyl (DEAE) dextran-mediated, lipid-mediated, cationic polymer-mediated like polyethyleneimine (PEI)-mediated and polylysine-mediated and polybrene-mediated, see, e.g., Chun Zhang et al., Polyethylenimine Strategies for Plasmid Delivery to Brain-Derived Cells, 33(2) Methods 144-150 (2004). Such chemical-mediated delivery systems can be prepared by standard methods and are commercially available, see, e.g., CELLPHECT Transfection Kit (Amersham Biosciences, Piscataway, N.J., USA); Mammalian Transfection Kit, Calcium phosphate and DEAE Dextran, (Stratagene, Inc.); LIPOFECTAMINE™ Transfection Reagent (Invitrogen, Inc., Carlsbad, Calif.); EXGEN 500 Transfection kit (Fermentas, Inc., Hanover, Md., USA), and SUPERFECT and EFFECTINE Transfection Kits (Qiagen, Inc., Valencia, Calif., USA).

In another aspect of this embodiment, a physical-mediated method is used to introduce a polynucleotide molecule encoding a modified Clostridial toxin or a fragment or variant thereof into a cell. Physical techniques include, without limitation, electroporation, biolistic and microinjection. Biolistics and microinjection techniques perforate the cell wall in order to introduce the nucleic acid molecule into the cell, see, e.g., Biewenga et al., J. Neurosci. Methods, 71(1), 67-75 (1997); and O'Brien et al., Methods 33(2), 121-125 (2004). Electroporation, also termed electropermeabilization, uses brief, high-voltage, electrical pulses to create transient pores in the membrane through which the nucleic acid molecules enter and can be used effectively for stable and transient transfections of all cell types, see, e.g., M. Golzio et al., 33(2) Methods 126-135 (2004); and Gresch et al., 33(2) Methods 151-163 (2004).

In another aspect of this embodiment, a viral-mediated method, termed transduction, is used to introduce a polynucleotide molecule encoding a modified Clostridial toxin or a fragment or variant thereof into a cell. In viral-mediated methods of transient transduction, the process by which viral particles infect and replicate in a host cell has been manipulated in order to use this mechanism to introduce a nucleic acid molecule into the cell. Viral-mediated methods have been developed from a wide variety of viruses including, without limitation, retroviruses, adenoviruses, adeno-associated viruses, herpes simplex viruses, picornaviruses, alphaviruses and baculoviruses, see, e.g., Blesch et al., 33(2) Methods 164-172 (2004); and Federico et al., 229 Methods Mol. Biol. 3-15 (2003); Poeschla et al., 5(5) Curr. Opin. Mol. Ther. 529-540 (2003); Benihoud et al., 10(5) Curr. Opin. Biotechnol. 440-447 (1999); Bueler et al., 380(6) Biol. Chem. 613-622 (1999); Lai et al., 21(12) DNA Cell Biol. 895-913 (2002); Burton et al., 21(12) DNA Cell Biol. 915-936 (2002); Grandi et al., 33(2) Methods 179-186 (2004); Frolov et al., 93(21) PNAS USA 11371-11377 (1996); Ehrengruber et al., 59(1) Brain Res. Bull. 13-22 (2002); Kost et al., 20(4) Trends Biotechnol. 173-180 (2002); and Huser et al., 3(1)Am. J. Pharmacogenomics 53-63 (2003).

Adenoviruses, which are non-enveloped, double-stranded DNA viruses, are often selected for mammalian cell transduction because adenoviruses handle relatively large polynucleotide molecules of about 36 kb, are produced at high titer, and can efficiently infect a wide variety of both dividing and non-dividing cells, see, e.g., Hermens et al., 71(1) J. Neurosci. Methods 85-98 (1997); and Mizuguchi et al., 52(3) Adv. Drug Deliv. Rev. 165-176 (2001). Transduction using adenoviral-based system do not support prolonged protein expression because the nucleic acid molecule is carried from an episome in the cell nucleus, rather than being integrated into the host cell chromosome. Adenoviral vector systems and specific protocols for how to use such vectors are disclosed in, e.g., VIRAPOWER™ Adenoviral Expression System (Invitrogen, Inc., Carlsbad, Calif., USA) and VIRAPOWER™ Adenoviral Expression System Instruction Manual 25-0543 version A, Invitrogen, Inc.; and ADEASY™ Adenoviral Vector System (Stratagene, Inc., La Jolla, Calif., USA) and ADEASY™ Adenoviral Vector System Instruction Manual, Stratagene, Inc.

Polynucleotide molecule delivery can also use single-stranded RNA retroviruses, such as, e.g., oncoretroviruses and lentiviruses. Retroviral-mediated transduction often produce transduction efficiencies close to 100%, can easily control the proviral copy number by varying the multiplicity of infection (MOI), and can be used to either transiently or stably transduce cells, see, e.g., Tonini et al., 285 Methods Mol. Biol. 141-148 (2004); Blesch et al., 33(2) Methods 164-172 (2004); Recillas-Targa et al., 267 Methods Mol. Biol. 417-433 (2004); and Wolkowicz et al., 246 Methods Mol. Biol. 391-411 (2004). Retroviral particles consist of an RNA genome packaged in a protein capsid, surrounded by a lipid envelope. The retrovirus infects a host cell by injecting its RNA into the cytoplasm along with the reverse transcriptase enzyme. The RNA template is then reverse transcribed into a linear, double stranded cDNA that replicates itself by integrating into the host cell genome. Viral particles are spread both vertically (from parent cell to daughter cells via the provirus) as well as horizontally (from cell to cell via virions). This replication strategy enables long-term persistent expression since the nucleic acid molecules of interest are stably integrated into a chromosome of the host cell, thereby enabling long-term expression of the protein. For instance, animal studies have shown that lentiviral vectors injected into a variety of tissues produced sustained protein expression for more than 1 year, see, e.g., Naldini et al., 272(5259) Science 263-267 (1996). The Oncoretroviruses-derived vector systems, such as, e.g., Moloney murine leukemia virus (MoMLV), are widely used and infect many different non-dividing cells. Lentiviruses can also infect many different cell types, including dividing and non-dividing cells and possess complex envelope proteins, which allows for highly specific cellular targeting.

Retroviral vectors and specific protocols for how to use such vectors are disclosed in, e.g., U.S. Pat. Nos. 5,464,758; 5,814,618; 5,514,578; 5,364,791; 5,874,534; and 5,935,934. Furthermore, such viral delivery systems can be prepared by standard methods and are commercially available, see, e.g., BD™ TET-OFF and TET-ON Gene Expression Systems (BD Biosciences-Clonetech, Palo Alto, Calif., USA) and BD™ TET-OFF and TET-ON Gene Expression Systems User Manual, BD Biosciences, GENESWITCH™ System (Invitrogen, Inc., Carlsbad, Calif., USA) and GENESWITCH™ System A Mifepristone-Regulated Expression System for Mammalian Cells version D, 25-0313, Invitrogen, Inc., (Nov. 4, 2002); VIRAPOWER™ Lentiviral Expression System (Invitrogen, Inc., Carlsbad, Calif., USA) and VIRAPOWER™ Lentiviral Expression System Instruction Manual, Invitrogen, Inc.; and COMPLETE CONTROL® Retroviral Inducible Mammalian Expression System (Stratagene, La Jolla, Calif., USA) and COMPLETE CONTROL® Retroviral Inducible Mammalian Expression System Instruction Manual.

The methods disclosed in the present specification include, in part, expressing a modified Clostridial toxin or a fragment or variant thereof from a polynucleotide molecule. It is envisioned that any of a variety of expression systems may be useful for expressing a modified Clostridial toxin or a fragment or variant thereof from a polynucleotide molecule disclosed in the present specification, including, without limitation, cell-based systems and cell-free expression systems. Cell-based systems include, without limitation, viral expression systems, prokaryotic expression systems, yeast expression systems, baculoviral expression systems, insect expression systems and mammalian expression systems. Cell-free systems include, without limitation, wheat germ extracts, rabbit reticulocyte extracts and E. coli extracts and generally are equivalent to the method disclosed herein. Expression of a polynucleotide molecule using an expression system can include any of a variety of characteristics including, without limitation, inducible expression, non-inducible expression, constitutive expression, viral-mediated expression, stably-integrated expression, and transient expression. Expression systems that include well-characterized vectors, reagents, conditions and cells are well-established and are readily available from commercial vendors that include, without limitation, Ambion, Inc., Austin, Tex.; BD Biosciences-Clontech, Palo Alto, Calif.; BD Biosciences Pharmingen, San Diego, Calif.; Invitrogen, Inc., Carlsbad, Calif.; QIAGEN, Inc., Valencia, Calif.; Roche Applied Science, Indianapolis, Ind.; and Stratagene, La Jolla, Calif. Non-limiting examples on the selection and use of appropriate heterologous expression systems are described in e.g., PROTEIN EXPRESSION. A PRACTICAL APPROACH (S. J. Higgins and B. David Hames eds., Oxford University Press, 1999); Joseph M. Fernandez & James P. Hoeffler, GENE EXPRESSION SYSTEMS. USING NATURE FOR THE ART OF EXPRESSION (Academic Press, 1999); and Rai et al., 80(9) Curr. Sci. 1121-1128, (2001). These protocols are routine procedures well within the scope of one skilled in the art and from the teaching herein.

A variety of cell-based expression procedures are useful for expressing a modified Clostridial toxin or a fragment or variant thereof encoded by polynucleotide molecule disclosed in the present specification. Examples included, without limitation, viral expression systems, prokaryotic expression systems, yeast expression systems, baculoviral expression systems, insect expression systems and mammalian expression systems. Viral expression systems include, without limitation, the VIRAPOWER™ Lentiviral (Invitrogen, Inc.) the Adenoviral Expression Systems (Invitrogen, Inc.), the ADEASY™ XL Adenoviral Vector System (Stratagene) and the VIRAPORT® Retroviral Gene Expression System (Stratagene). Non-limiting examples of prokaryotic expression systems include the CHAMPION™ pET Expression System (EMD Biosciences-Novagen, Madison, Wis., USA), the TRIEX™ Bacterial Expression Systems (EMD Biosciences-Novagen, Madison, Wis., USA), the QIAEXPRESS® Expression System (QIAGEN, Inc.), and the AFFINITY® Protein Expression and Purification System (Stratagene). Yeast expression systems include, without limitation, the EASYSELECT™ Pichia Expression Kit (Invitrogen, Inc.), the YES-ECHO™ Expression Vector Kits (Invitrogen, Inc.) and the SPECTRA™ S. pombe Expression System (Invitrogen, Inc., Carlsbad, Calif.). Non-limiting examples of baculoviral expression systems include the BACULODIRECT™ (Invitrogen, Inc.), the BAC-TO-BAC® (Invitrogen, Inc.), and the BD BACULOGOLD™ (BD Biosciences-Pharmigen, San Diego, Calif., USA). Insect expression systems include, without limitation, the Drosophila Expression System (DES®) (Invitrogen, Inc., Carlsbad, Calif.), INSECTSELECT™ System (Invitrogen, Inc.) and INSECTDIRECT™ System (EMD Biosciences-Novagen). Non-limiting examples of mammalian expression systems include the T-REX™ (Tetracycline-Regulated Expression) System (Invitrogen, Inc.), the FLP-IN™ T-REx™ System (Invitrogen, Inc.), the PCDNA™ system (Invitrogen, Inc.), the pSecTag2 system (Invitrogen, Inc.), the EXCHANGER® System, INTERPLAY™ Mammalian TAP System (Stratagene), COMPLETE CONTROL® Inducible Mammalian Expression System (Stratagene) and LAC SWITCH® II Inducible Mammalian Expression System (Stratagene).

Another procedure of expressing a modified Clostridial toxin or a fragment or variant thereof encoded by polynucleotide molecule disclosed in the present specification employs a cell-free expression system such as, without limitation, prokaryotic extracts and eukaryotic extracts. Non-limiting examples of prokaryotic cell extracts include the RTS 100 E. coli HY Kit (Roche Applied Science, Indianapolis, Ind., USA), the ACTIVEPRO In vitro Translation Kit (Ambion, Inc., Austin, Tex., USA), the ECOPRO™ System (EMD Biosciences) and the EXPRESSWAY™ Plus Expression System (Invitrogen, Inc.). Eukaryotic cell extract include, without limitation, the RTS 100 Wheat Germ CECF Kit (Roche Applied Science, Indianapolis, Ind., USA), the TNT® Coupled Wheat Germ Extract Systems (Promega Corp.), the Wheat Germ IVT™ Kit (Ambion, Inc.), the Retic Lysate IVT™ Kit (Ambion, Inc.), the PROTEINSCRIPT® II System (Ambion, Inc.) and the TNT® Coupled Reticulocyte Lysate Systems (Promega Corp.).

Codon Optimized Sequences

Included herein are codon-optimized sequences of the aforementioned nucleic acid sequences and vectors. Codon optimization for expression in a host cell, e.g., bacteria such as E. coli or insect Hi5 cells, may be performed using Codon Optimization Tool (CODONOPT), available freely from Integrated DNA Technologies, Inc., Coralville, Iowa, USA.

Compositions

Embodiments of the disclosure further relate to compositions containing one or more Clostridial toxins, including, fragments or variants thereof and a carrier. Further embodiments relate to compositions comprising nucleic acids, codon-optimized nucleic acids, vectors, and production systems, e.g., host cells, encoding one or more Clostridial toxins, including, fragments or variants thereof. Still further, embodiments of the disclosure relate to compositions comprising antibodies which bind with specificity to one or more Clostridial toxins, including, fragments or variants thereof.

In one aspect, the disclosure relates to a pharmaceutical composition which is pharmaceutically acceptable. As used herein, the term “pharmaceutically acceptable” refers to any molecular entity or composition that does not produce an adverse, allergic, or other untoward or unwanted reaction when administered to an individual. As used herein, the term “pharmaceutically acceptable composition” is synonymous with “pharmaceutical composition” and refers to a therapeutically effective concentration of an active ingredient, such as, e.g., any of the Clostridial toxins, fragments, variants, or chimeras disclosed in the present specification. A pharmaceutical composition comprising a Clostridial toxin or Clostridial toxin fragment or a variant thereof is useful for medical and veterinary applications. A pharmaceutical composition may be administered to a patient alone, or in combination with other supplementary active ingredients, agents, drugs or hormones. The pharmaceutical compositions may be manufactured using any of a variety of processes, including, without limitation, conventional mixing, dissolving, granulating, levigating, emulsifying, encapsulating, entrapping, and lyophilizing. The pharmaceutical composition can take any of a variety of forms including, without limitation, a sterile solution, suspension, emulsion, lyophilizate, tablet, pill, pellet, capsule, powder, syrup, elixir or any other dosage form suitable for administration.

It is also envisioned that a pharmaceutical composition comprising a Clostridial toxin or Clostridial toxin fragment or variant disclosed in the present specification can optionally include pharmaceutically acceptable carriers that facilitate processing of an active ingredient into pharmaceutically acceptable compositions. As used herein, the term “pharmaceutically acceptable carrier” and refers to any carrier that has substantially no long term or permanent detrimental effect when administered and encompasses terms such as “pharmaceutically acceptable vehicle, stabilizer, diluent, additive, auxiliary, or excipient.” Such a carrier generally is mixed with an active compound or is permitted to dilute or enclose the active compound and can be a solid, semi-solid, or liquid agent. It is understood that the active ingredients can be soluble or can be delivered as a suspension in the desired carrier or diluent. Any of a variety of pharmaceutically acceptable carriers can be used including, without limitation, aqueous media such as, e.g., water, saline, glycine, hyaluronic acid and the like; solid carriers such as, e.g., mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like; solvents; dispersion media; coatings; antibacterial and antifungal agents; isotonic and absorption delaying agents; or any other inactive ingredient. Selection of a pharmacologically acceptable carrier can depend on the mode of administration. Except insofar as any pharmacologically acceptable carrier is incompatible with the active ingredient, its use in pharmaceutically acceptable compositions is contemplated. Non-limiting examples of specific uses of such pharmaceutical carriers can be found in PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS (Howard C. Ansel et al., eds., Lippincott Williams & Wilkins Publishers, 7th ed. 1999); REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY (Alfonso R. Gennaro ed., Lippincott, Williams & Wilkins, 20th ed. 2000); GOODMAN & GILMAN'S THE PHARMACOLOGICAL BASIS OF THERAPEUTICS (Joel G. Hardman et al., eds., McGraw-Hill Professional, 10th ed. 2001); and HANDBOOK OF PHARMACEUTICAL EXCIPIENTS (Raymond C. Rowe et al., APhA Publications, 4th edition 2003). These protocols are routine procedures and any modifications are well within the scope of one skilled in the art and from the teaching herein.

It is further envisioned that a pharmaceutical composition disclosed in the present specification can optionally include, without limitation, other pharmaceutically acceptable components (or pharmaceutical components), including, without limitation, buffers, preservatives, tonicity adjusters, salts, antioxidants, osmolality adjusting agents, physiological substances, pharmacological substances, bulking agents, emulsifying agents, wetting agents, sweetening or flavoring agents, and the like. Various buffers and refers to for adjusting pH can be used to prepare a pharmaceutical composition disclosed in the present specification, provided that the resulting preparation is pharmaceutically acceptable. Such buffers include, without limitation, acetate buffers, citrate buffers, phosphate buffers, neutral buffered saline, phosphate buffered saline and borate buffers. It is understood that acids or bases can be used to adjust the pH of a composition as needed. Pharmaceutically acceptable antioxidants include, without limitation, sodium metabisulfite, sodium thiosulfate, acetylcysteine, butylated hydroxyanisole and butylated hydroxytoluene. Useful preservatives include, without limitation, benzalkonium chloride, chlorobutanol, thimerosal, phenylmercuric acetate, phenylmercuric nitrate, a stabilized oxy chloro composition, such as, e.g., PURITE® and chelants, such as, e.g., DTPA or DTPA-bisamide, calcium DTPA, and CaNaDTPA-bisamide. Tonicity adjustors useful in a pharmaceutical composition include, without limitation, salts such as, e.g., sodium chloride, potassium chloride, mannitol or glycerin and other pharmaceutically acceptable tonicity adjustor. The pharmaceutical composition may be provided as a salt and can be formed with many different acids, including, but not limited to, hydrochloric, sulfuric, acetic, lactic, tartaric, malic, and succinic. Salts tend to be more soluble in aqueous or other protonic solvents than are the corresponding free base forms. It is understood that these and other substances known in the art of pharmacology can be included in a pharmaceutical composition useful in the specification.

In an embodiment, a composition comprises a Clostridial toxin or Clostridial toxin fragment or variant and a viscous carrier. “Viscous carrier” means a biocompatible compound which when formulated with a botulinum neurotoxin provides upon in vivo local injection of the formulation a depot from which the Clostridial toxin or a fragment or variant thereof is released in amounts such that the extent of diffusion of the Clostridial toxin or a fragment or variant thereof away from the site of the local injection and/or the amount of the Clostridial toxin or a fragment or variant thereof which diffuses away from the site of local injection is significantly reduced. Any suitable viscous carrier, for example, ophthalmically acceptable viscous carrier, may be employed in accordance with the present disclosure. The viscous carrier is present in an amount effective in providing the desired viscosity to the drug delivery system. Advantageously, the viscous carrier is present in an amount in a range of from about 0.5 wt % to about 95 wt % of the drug delivery system. The specific amount of the viscous carrier used depends upon a number of factors including, for example and without limitation, the specific viscous carrier used, the molecular weight of the viscous carrier used, the viscosity desired for the present drug delivery system being produced and/or used and like factors.

Examples of useful viscous carriers include, but are not limited to, hyaluronic acid, carbomers, polyacrylic acid, cellulosic derivatives, polycarbophil, polyvinylpyrrolidone, gelatin, dextrin, polysaccharides, polyacrylamide, polyvinyl alcohol, polyvinyl acetate, heparin, proteoglycan (HSPG), heparin sulfate (HS), derivatives thereof and mixtures thereof. Representative types of viscous carriers are disclosed in U.S. Pat. Nos. 9,044,477; 9,622,957; 9,050,336.

A dermal filler can also be used as the viscous carrier. Suitable dermal fillers for that purpose include collagen (sterile collagen is sold under the trade names ZYDERM, ZYPLAST, COSMODERM, COSMOPLAST and AUTOLGEN), HYLAFORM® (hyaluronic acid), RESTYLANE® (hyaluronic acid), SCULPTRA™ (polylactic acid), RADIESSE™ (calcium hydroxyl apatite) and JUVEDERM™. JUVEDERM™, available from Allergan, Inc. (Irvine, Calif., USA) comprises a sterile, biodegradable, non-pyrogenic, viscoelastic, clear, colorless, homogenized gel consisting of cross-linked hyaluronic acid formulated at a concentration of 24 mg/ml in a physiologic buffer. Representative types of dermal fillers are disclosed in U.S. Pat. Nos. 9,622,957; 9,161,970; 9,050,336.

The molecular weight of the presently useful viscous carrier can be in a range of about 10,000 Daltons or less to about 2 million Daltons or more. In one particularly useful embodiment, the molecular weight of the viscous carrier is in a range of about 100,000 Daltons or about 200,000 Daltons to about 1 million Daltons or about 1.5 million Daltons. Again, the molecular weight of the viscous carrier useful in accordance with the present disclosure, may vary over a substantial range based on the type of viscous carrier employed, and the desired final viscosity of the present drug delivery system in question, as well as, possibly other factors.

In one very useful embodiment, the carrier is a polymeric hyaluronate component, for example, a metal hyaluronate component, preferably selected from alkali metal hyaluronates, alkaline earth metal hyaluronates and mixtures thereof, and still more preferably selected from sodium hyaluronates, and mixtures thereof. The molecular weight of such hyaluronate component preferably is in a range of about 50,000 Daltons or about 100,000 Daltons to about 1.3 million Daltons or about 2 million Daltons. In one embodiment, the present compositions include a polymeric hyaluronate component in an amount in a range about 0.05% to about 0.5% (w/v). In a further useful embodiment, the hyaluronate component is present in an amount in a range of about 1% to about 4% (w/v) of the composition. In this latter case, the very high polymer viscosity forms a gel that slows particle sedimentation rate to the extent that often no resuspension processing is necessary over the estimated shelf life, for example, at least about 2 years, of the drug delivery system. Such a drug delivery system can be marketed in pre-filled syringes since the gel cannot be easily removed by a needle and syringe from a bulk container.

In another embodiment, the carrier is a thermo-reversible gelling agent, such as, e.g., poloxamer-407, which is described in U.S. Pat. No. 9,107,815. Such compositions comprising thermo-reversible gels can be administered (as by injection) as a low viscosity liquid that rapidly increases in viscosity after injection. The resulting high viscosity matrix is adhesive, biodegradable and biocompatible and upon administration forms a depot from which the botulinum toxin can be released, thereby providing a sustained or extended release drug delivery system. In this manner, a lower dose of the botulinum toxin can be used. Such a pharmaceutical composition can be administered pre-mixed or as a simple reconstitution vehicle or its several compartments combined at the time of administration, as by use of a dual chamber syringe. Representative types of thermo-reversible gelling agents are disclosed in, e.g., U.S. Pat. Nos. 8,168,206; 8,642,047; and 9,278,140.

In some embodiments, to increase the resident time of the Clostridial toxin or a fragment or variant thereof in the joint, the Clostridial toxin or a fragment or variant thereof is provided in a controlled release system comprising a polymeric matrix encapsulating the Clostridial toxin or a fragment or variant thereof, wherein fractional amount of the Clostridial toxin or a fragment or variant thereof is released from the polymeric matrix over a prolonged period of time in a controlled manner. Controlled release neurotoxin systems have been disclosed, for example, in U.S. Pat. Nos. 6,585,993; 6,585,993; 6,306,423; and 6,312,708.

In one embodiment, the disclosure relates to topical compositions comprising a Clostridial toxin or a fragment or variant thereof. Representative examples include, e.g., a topical cream comprising BoNT/A fragments, e.g., the full-length heavy chain (H_(C)) or the N-terminal domain thereof (H_(CN)) of Clostridial toxins, or variants thereof, which are delivered via commercially viable ionic nanoparticle technology, INPART® (Transdermal Corp., Birmingham, Mich., USA). See U.S. Pat. Nos. 7,838,011; 7,727,537; 8,568,740.

Pharmaceutical compounds and formulations for topical administration may include ointments, lotions (e.g., skin care lotion), creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be used. Preferred topical formulations include those in which the compounds of the invention are in admixture with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants. Preferred lipids and liposomes include neutral (e.g., dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline) negative (e.g., dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g., dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidyl ethanolamine DOTMA). The Clostridial toxins of the disclosure or fragments or variants thereof may be encapsulated within liposomes or may form complexes thereto, in particular to cationic liposomes. Alternatively, compounds may be complexed to lipids, in particular to cationic lipids. Preferred fatty acids and esters include but are not limited arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a C₁₋₁₀ alkyl ester (e.g., isopropylmyristate IPM), monoglyceride, diglyceride or pharmaceutically acceptable salt thereof. Topical formulations are described in detail in U.S. Pat. No. 6,747,014.

In another embodiment, the disclosure relates to transdermal compositions comprising a Clostridial toxin or a fragment or variant thereof, more specifically to such compositions that enable the transport or delivery of a Clostridial toxin or a fragment or variant thereof through the skin or epithelium (also referred to as “transdermal delivery”). Such compositions may be used as topical applications for providing a botulinum toxin to a subject, for various therapeutic, aesthetic and/or cosmetic purposes, as described herein. For instance, the composition for topical delivery may comprise a positively charged carrier molecule having efficiency groups, such that the toxin is administered transdermally to muscles and/or other skin-associated structures. The transport occurs without covalent modification of the botulinum toxin. Exemplary compositions and delivery systems are provided in patches developed by Revance Therapeutics, e.g., U.S. Pat. Nos. 8,568,740; 8,518,414; 9,180,081; 8,404,249; 8,962,548; 9,211,248; 8,398,997; 8,974,774; 8,926,991; and 8,092,788. See also U.S. Pat. Nos. 8,404,249; 9,144,692; and 7,704,524. In one embodiment, the transdermal delivery system is a patch. Transdermal patches are generally characterized as having an adhesive layer, which will be applied to a person's skin, a depot or reservoir for holding a pharmaceutical agent, and an exterior surface that prevents leakage of the pharmaceutical from the depot. The exterior surface of a patch is typically non-adhesive. In accordance with the present disclosure, the Clostridial toxin or a fragment or variant thereof is incorporated into the patch so that the neurotoxin remains stable for extended periods of time. The Clostridial toxin or a fragment or variant thereof may be incorporated into a polymeric matrix that stabilizes the Clostridial toxin or a fragment or variant thereof, and permits the Clostridial toxin or a fragment or variant thereof to diffuse from the matrix and the patch. In one embodiment of the disclosure, the composition containing the toxin and the enhancing agent is provided in an adhesive patch. The Clostridial toxin or a fragment or variant thereof may also be incorporated into the adhesive layer of the patch so that once the patch is applied to the skin, the Clostridial toxin or a fragment or variant thereof may diffuse through the skin. Examples of adhesive patches for the delivery of proteins are well known. For example, see U.S. Pat. No. 296,006 (design patent); U.S. Pat. Nos. 6,010,715; 5,591,767; 5,008,110; 5,683,712; 5,948,433; and 5,965,154. In some embodiments, the patches may include lipid vesicles (see, U.S. Pat. No. 6,165,500). In some embodiments, the patches may include stinging cells (specifically, cnidocytes, nematocytes, or ptychocytes) of an organism belonging to the phylum Cnidaria (e.g., Aiptasia sp.) which have been transformed with a vector comprising a nucleic acid encoding the Clostridial toxin of the disclosure or a fragment or variant thereof. In some embodiments, the patches may include a transgenic organism of the phylum Cnidaria which expresses the Clostridial toxin of the disclosure or a fragment or variant thereof in specialized cells, e.g., stinging cells.

Kits

Another aspect of the disclosure relates to a kit comprising a Clostridial toxin or a fragment or variant thereof, including nucleic acids encoding the Clostridial toxin or a fragment or variant thereof, or an antibody binding to the Clostridial toxin or a fragment or variant thereof and an instructional material. In one embodiment, the Clostridial toxin or a fragment or variant thereof is part of an immunogenic composition. In another embodiment, the Clostridial toxin or a fragment or variant thereof is part of a conjugate, e.g., a tagged protein. As used herein, an “instructional material” includes a publication, a recording, a diagram, or any other medium of expression which is used to communicate the usefulness of the Clostridial toxin or a fragment or variant thereof for diagnosing, imaging, treating, ameliorating, relieving, inhibiting, preventing, or reducing a disorder in a subject or for administering such a composition via a route described herein. The instructional material may also, for example, describe an appropriate dose of the Clostridial toxin or a fragment or variant thereof. The instructional material of the kit of the disclosure may, for example, be affixed to a container which contains a Clostridial toxin or a fragment or variant thereof or be shipped together with a container which contains the Clostridial toxin or a fragment or variant thereof. Alternatively, the instructional material may be shipped separately from the container with the intention that the instructional material and the modified BoNT/A polypeptide be used cooperatively by the recipient.

The disclosure also includes a kit comprising Clostridial toxin or a fragment or variant thereof and a delivery device for delivering the polypeptide to a subject. By way of example, the delivery device may be a squeezable spray bottle, a metered-dose spray bottle, an aerosol spray device, an atomizer, a dry powder delivery device, a self-propelling solvent/powder dispensing device, a syringe, a needle, a tampon, or a dosage measuring container. The kit may further comprise an instructional material as described herein.

Typically, the container may hold one or more formulations and a label on, or associated with, the container that may indicate directions for reconstitution and/or use. For example, the label may indicate that the formulation is reconstituted to concentrations as described above. The label may further indicate that the formulation is useful or intended for, for example, cutaneous administration. In some embodiments, a container may contain a single dose of a stable formulation containing the Clostridial toxin or a fragment or variant thereof. In various embodiments, a single dose of the stable formulation is present in a volume of less than about 0.5 ml or less. Alternatively, a container holding the formulation may be a multi-use vial, which allows for repeat administrations (e.g., from 2-6 administrations) of the formulation. Kits or other articles of manufacture may further include a second container comprising a suitable diluent (e.g., BWFI, saline, buffered saline). Upon mixing of the diluent and the formulation, the final polypeptide concentration in the reconstituted formulation will generally be at least 1 pg/ml (e.g., at least 5 pg/ml, at least 10 pg/ml, at least 20 pg/ml, at least 50 pg/ml, at least 100 pg/ml, at least 300 pg/ml, at least 500 pg/ml, at least 1 ng/ml, at least 3 ng/ml, at least 10 ng/ml, 0.1 μg/ml, 0.3 μg/ml, 1 μg/ml, 3 μg/ml, 10 μg/ml, 30 μg/ml, 100 μg/ml, or more). Kits or other articles of manufacture may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. In some embodiments, kits or other articles of manufacture may include an instruction for administration.

Devices and Systems

Embodiments of the present disclosure further relate to devices and/or systems comprising the Clostridial toxins, including, fragments or variants thereof. Representative examples of delivery systems include, e.g., polyetherester copolymer microspheres for encapsulation and controlled delivery of a variety of protein drugs, including tetanus and botulinum antitoxins (see, U.S. Pat. No. 5,980,948); microspherical particles comprising a continuous matrix of biodegradable polymer containing discrete regions containing botulinum toxins (U.S. Pat. No. 5,902,565). In another embodiment, the delivery systems include implants for pulsatile or continuous in vivo release of a neurotoxin over a period ranging from several days to a few years (see, e.g., U.S. Pat. Nos. 6,383,509; 6,506,399; 6,312,708; 6,585,993; and 6,306,423). As used herein, “implant” generally relates to a controlled release (e.g., pulsatile or continuous) composition or drug delivery system. The implant can be, for example, injected, inserted or implanted into a subject's body. The implant may be administered for a few days up to a year or more, e.g., 7 days, 15 days, 30 days, 1 month, 3 months, 6 months, 1 year, or more.

In one specific embodiment, provided herein are delivery systems for delivering the Clostrodial toxins into the skin of a subject. Human skin has two distinct layers and varies in thickness from about 1.5 to about 4 mm or more, depending on the regions of the body. The first layer is the superficial layer called the epidermis. It is a relatively thick epithelium. Deep to the epidermis is the second layer called the dermis. The dermis is a fibrous connective tissue and comprises sweat glands and nerves, or nerve terminals, innervating such sweat glands. Just below the skin lies a fatty layer called the hypodermis, which may also be considered a part of a subcutaneous layer. Beneath the hypodermis or subcutaneous layer lies the deep fascial investment of the specialized structures of the body, for example the muscles.

Accordingly, in one embodiment the delivery delivers a Clostrodial toxin, or DNA encoding the Clostrodial toxin, to a tissue of an animal or a human subject. In one embodiment, the Clostrodial toxin is delivered to the layer of the skin in which nerve terminals are found. For example, delivery is to the dermis layer. In another embodiment, delivery is to at least one layer of the skin and substantially to tissues beneath. For example, the Clostrodial toxin or a fragment or variant thereof is delivered to the dermis layer of the skin and to the subcutaneous layer. In another embodiment, the Clostrodial toxin is delivered to the skin and to muscle tissues beneath. In still another embodiment, Clostrodial toxin is delivered substantially to the muscle tissue.

The delivery of a composition comprising a carrier and a Clostrodial toxin and/or DNA encoding a Clostrodial toxin to a site may be accomplished via any means known in the art, e.g., needle-based delivery methods or needle-less delivery methods.

In one embodiment, the compositions are delivered via needleless delivery. Needleless injectors and their use are well known in the art. See for example, U.S. Pat. Nos. 6,053,889; 6,013,050; 6,010,478; 6,004,286 and 5,899,880, which disclose needleless injectors. In one embodiment, the needleless injector comprises an elongated tubular nozzle and is connected to or capable of connection to a suitable energizing means for producing a supersonic gas flow, for example a burst of helium, which accelerates mediums to high velocity toward a skin surface and into the skin surface. Such a device may be purchased from POWDERJECT Pharmaceuticals, Oxford, UK. In one embodiment, the gas pressure provided must be sufficient to discharge the compositions into a targeted site, for example the dermis, but not so great as to damage the target. In another embodiment, the gas pressure provided is sufficient to deliver the compositions to a target site, for example the dermis, but not so great as to damage the skin surface, for example the epithelium. In another embodiment, the gas pressure is sufficient to deliver the compositions to the dermis layer, but not to the layers below, for example the subcutaneous layer and/or the muscle tissues. In another embodiment, the gas pressure provided must be sufficient to discharge the drug particles into a targeted site, for example the dermis and/or substantially to the muscle tissue below, but not so great as to damage the skin surface.

Advantages for using a needleless injector include, for example, an optimal delivery to a specific tissue layer, for example the dermis layer. Furthermore, in the case where the delivery is to the dermis and not the muscle tissues, the treatment may not cause a loss of motor function in the area being treated. Also, the use of a needleless injector improves clinical safety by eliminating the risk of infection from accidental injury with needles or from potential splash back of bodily fluids from liquid jet injectors, thereby avoiding the possibilities of cross-contamination of blood-borne pathogens such as HIV and hepatitis B. The needleless injector also offers an optimal and specific delivery of drug particles to treat conditions with little pain or skin damage such as bruising or bleeding. Needless systems containing purified Clostrodial toxins are disclosed in U.S. Pat. No. 7,255,865.

In another embodiment, the disclosure further relates to needle-based systems comprising Clostrodial toxins and a carrier. Representative types of injection systems are known in the art, e.g., U.S. Pat. No. 8,603,028 (relating to injection devices having an angled tip portion); U.S. Pat. No. 8,801,659 (relating to injection devices for delivering viscous agents into soft tissues). In certain embodiments, compositions can be injected into a site via traditional delivery systems, e.g., syringes, catheters, needles and other devices.

Dosages

In one embodiment, the compositions disclosed herein contain an effective amount of Clostrodial toxin or a fragment thereof. The term “effective amount,” when used with respect to treating a condition, can be a dose sufficient to treat the symptoms, for example, by at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%. In the context of the Clostrodial toxins comprising only the binding domains (and/or fragments thereof, the dose generally is in the range of 0.1-1000 mg/day and can be, for example, in the range of 1-500 pg/day, 100-5000 pg/day, 1-500 ng/day, 100-5000 ng/day, 1-500 μg/day, 100-5000 μg/day, 5-1000 mg/day, 10-500 mg/day, 20-500 mg/day, 50-500 mg/day, 10-200 mg/day, 10-100 mg/day or 100-500 mg/day, with the actual amount to be administered determined by a physician taking into account the relevant circumstances including the age and weight of the patient, the patient's general physical condition, and the route of administration. Where repeated administration is used, the frequency of administration depends, in part, on the half-life of the pharmaceutical composition.

In another embodiment, the concentration of the Clostridial toxin or a fragment or variant thereof in the formulation can be in the range of about 1 pg/ml to 1000 pg/ml toxin, 1 ng/ml to 1000 μg/ml toxin, for example, about 10 ng/ml to 500 μg/ml toxin, 100 ng/ml to 100 μg/ml toxin, 200 ng/ml to 500 μg/ml toxin, 200 ng/ml to 5000 ng/ml toxin, 500 ng/ml to 5000 ng/ml, 10 ng/ml to 5000 ng/ml, 20 ng/ml to 5000 ng/ml, 50 ng/ml to 1000 ng/ml, 100 ng/ml to 10 μg/ml, 100 ng/ml to 5000 ng/ml, 100 ng/ml to 1000 ng/ml, 10 ng/ml to 100 ng/ml, 200 ng/ml to 10 μg/ml, 200 ng/ml to 1000 ng/ml, 500 ng/ml to 50 μg/ml, 500 ng/ml to 10 μg/ml or 1000 ng/ml to 10 μg/ml toxin. In another embodiment, the concentration of the Clostridial toxin or a fragment or variant thereof in the formulation can be in the range of about 1 pM to 500 pM, 0.1 nM to 500 μM, 1.0 nM to 500 μM, 1.0 nM to 100 μM, 1.0 nM to 50 μM, 1.0 nM to 10 μM, 1.0 nM to 500 nM, 1.0 nM to 100 nM, 1.0 nM to 10 nM, 10 nM to 100 μM, 10 nM to 50 μM, 10 nM to 10 μM, 10 nM to 5 μM, 10 nM to 1 μM, 50 nM to 500 μM, 50 nM to 100 μM, 50 nM to 10 μM, 50 nM to 1 μM, 50 nM to 500 nM, 50 nM to 100 nM, 1 nM to 10 μM, 10 nM to 10 μM, 20 nM to 10 μM, 100 nM to 10 μM, 1 μM to 1 mM, 1 μM to 500 μM, 1 μM to 500 μM, 5 μM to 500 μM, 10 μM to 100 μM, 1 nM to 500 μM, 10 nM to 100 μM, 10 nM to 10 μM, 20 nM to 1 μM, 1 nM to 500 nM, 10 nM to 100 nM, 20 nM to 50 nM, 1 nM to 100 nM, 3 nM to 50 nM toxin.

Dosing can be single dosage or cumulative (serial dosing), and can be readily determined by one skilled in the art. For instance, treatment of a dermal disorder may comprise a one-time administration of an effective dose of a composition disclosed herein. As a non-limiting example, an effective dose of a composition disclosed herein can be administered once to an individual, e.g., as a single injection or deposition at or near the site exhibiting a symptom of a cosmetic disorder. Alternatively, treatment of a cosmetic disorder may comprise multiple administrations of an effective dose of a composition disclosed herein carried out over a range of time periods, such as, e.g., daily, once every few days, weekly, monthly or yearly. As a non-limiting example, a composition disclosed herein can be administered once or twice yearly to an individual. The timing of administration can vary from individual to individual, depending upon such factors as the severity of an individual's symptoms. For example, an effective dose of a composition disclosed herein can be administered to an individual once a month for an indefinite period of time, or until the individual no longer requires therapy. A person of ordinary skill in the art will recognize that the condition of the individual can be monitored throughout the course of treatment and that the effective amount of a composition disclosed herein that is administered can be adjusted accordingly.

Routes of Administration

An active agent (e.g., a Clostridial toxin or a fragment or variant thereof) is administered to an individual using any available method and route suitable for drug delivery, including in vivo and ex vivo methods, as well as systemic and localized routes of administration. In one embodiment, the Clostridial toxin, including fragments or variants thereof, are administered in accordance with established protocols for botulinum toxin therapy. The term “botulinum toxin therapy” encompasses, without limitation, the use of any naturally occurring or modified or engineered form of a botulinum toxin or a domain or fragment thereof, in any formulation, combined with any carrier or active ingredient and administered by any route of administration.

Methods of Use

Therapeutic Applications

In one embodiment, the present disclosure relates to methods for using Clostridial toxins or fragments or variants thereof for therapeutic applications. In another embodiment, the compositions are useful in treating and/or reducing the occurrence of a disease or disorder associated with ECM dysregulation. In one embodiment, the compositions are useful in treating and/or reducing the occurrence of a fibrotic disorder or a fibrotic disease. In another embodiment, the compositions are useful in treating and/or reducing the occurrence of a disease or disorder associated with connective tissue fibroblasts.

In some embodiments, the present disclosure relates to treatment of a disease selected from extracellular matrix disease; connective tissue disease; endothelial diseases; and fibrotic diseases comprising administering to a subject in need thereof, a therapeutically effective amount of a composition comprising a polypeptide having an amino acid sequence with at least about 90% sequence identity to a binding domain of a botulinum toxin, wherein the molecular weight of the polypeptide is between about 4 kDa to about 60 kDa; preferably between about 10 kDa to about 60 kD; particularly between about 12 kDa to about 50 kDa.

In some embodiments, the therapy is carried out by administering a botulinum toxin fragment of BoNT/A, BoNT/B, BoNT/C₁, BoNT/D, BoNT/E, BoNT/F, BoNT/FA, BoNT/G, BoNT/H, BoNT/DC, BoNT/CD, BoNT/X, or eBoNT/J, wherein the fragment has a molecular weight between about 4 kDa to about 60 kDa; preferably between about 10 kDa to about 60 kD; particularly between about 12 kDa to about 50 kDa.

In some embodiments, the therapy is carried out by administering a botulinum toxin polypeptide comprising an amino acid sequence having at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or greater %, e.g., 99% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 19, SEQ ID NOs: 3-5, SEQ ID NO: 6, SEQ ID NO: 20, SEQ ID Nos: 7-10, SEQ ID NO: 11, SEQ ID NO: 21, SEQ ID NOs: 12-18 and SEQ ID NOs: 25-27.

In some embodiments, the therapy is carried out by administering a botulinum toxin polypeptide comprising an amino acid sequence comprising, consisting essentially of, or consisting of a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 19, SEQ ID NOs: 3-5, SEQ ID NO: 6, SEQ ID NO: 20, SEQ ID Nos: 7-10, SEQ ID NO: 11, SEQ ID NO: 21, SEQ ID NOs: 12-18 and SEQ ID NOs: 25-27.

In some embodiments, the present disclosure relates to treatment of an extracellular matrix disease, e.g., atherosclerosis, cancer, amyloid diseases, glomerular diseases, mesangial diseases, inflammatory conditions, and developmental disorders, comprising administering to a subject in need thereof, a therapeutically effective amount of a composition comprising a binding domain of a botulinum toxin, as provided above.

In some embodiments, the present disclosure relates to treatment of a connective tissue disease, e.g., systemic lupus erythematosus (SLE), rheumatoid arthritis, systemic sclerosis, Sjörgen's syndrome, mixed connective tissue disease, eosinophilia infectiosa, polymyositis, dermatomysistis, periarteriitis nodosa, Wegener-granulomatose, CREST-syndrome and SHARP-syndrome; preferably systemic lupus erythematosus (SLE), rheumatoid arthritis, systemic sclerosis, or Sjörgen's syndrome, comprising administering to a subject in need thereof, a therapeutically effective amount of a composition comprising a binding domain of a botulinum toxin, as provided above.

In some embodiments, the present disclosure relates to treatment of an endothelial disease, e.g., Alzheimer's disease, amyotrophic lateral sclerosis, diabetic neuropathy, stroke, atherosclerosis, diabetes, restenosis, coronary artery disease, peripheral vascular disease, vascular leak, vasculitis, vasculitidis, Wegner's disease, gastric or oral ulcerations, cirrhosis, hepatorenal syndrome, Crohn's disease, hair loss, skin purpura, telangiectasia, venous lake formation, delayed wound healing, pre-eclampsia, sepsis, ischemia-reperfusion injury, hypertension, chronic or acute infection, menorrhagia, neonatal respiratory distress, pulmonary fibrosis, emphysema, nephropathy, glomerulonephritis, sclerodoma, and vascular abnormalities or an endothelial disease characterized by insufficient angiogenesis, hypertension, vasoconstriction, vascular leak, altered vasomotor tone, hypercoagulation, anti-inflammatory properties, or poor endothelial cell health, comprising administering to a subject in need thereof, a therapeutically effective amount of a composition comprising a binding domain of a botulinum toxin, as provided above.

In some embodiments, the present disclosure relates to treatment of a fibrotic disease, e.g., dermal scars, wound scars, surgical scars, hypertrophic scarring, keloid, glial scar, postoperative intraperitoneal adhesions, fibroma, liver fibrosis, cirrhosis, pancreatitis, benign prostatic hyperplasia, breast fibrosis, retroperitoneal fibrosis, e.g., fibrotic bladder, interstitial cystitis, pulmonary fibrosis (e.g., idiopathic pulmonary fibrosis), vascular fibrosis (e.g., atherosclerosis, stenosis, restenosis), kidney fibrosis, glomerulosclerosis, atrial fibrosis, cardiac fibrosis, endomyocardial fibrosis, arterial stiffness, arthrofibrosis, mediastinal fibrosis, myelofibrosis, progressive massive fibrosis (e.g., lungs), chronic kidney disease (CKD), nephrogenic systemic fibrosis, Crohn's disease, scleroderma, systemic sclerosis (e.g., skin, lungs), athrofibrosis (e.g., knee, shoulder, other joints), Peyronie's disease, Dupuytren's contracture, adhesive capsulitis, organ transplant associated fibrosis, ischemia associated fibrosis, cystic fibrosis, non-cystic fibrosis, mixed connective tissue disease, ocular fibrosis, osteoarthritis, scleroderma, or asthma, comprising administering to a subject in need thereof, a therapeutically effective amount of a composition comprising a binding domain of a botulinum toxin, as provided above. Preferably, the disclosure relates to treatment of a fibrotic disease selected from fibrotic breast disease; keloid scar; liver fibrosis; fibrotic bladder; hyperplasia of fibrous tissue, e.g., benign prostatic hyperplasia; pancreatitis; adhesion, e.g., intra-abdominal adhesion; scarring, e.g., hypertrophic scars or a combination thereof, comprising administering to a subject in need thereof, a therapeutically effective amount of a composition comprising a binding domain of a botulinum toxin, as provided above.

In some embodiments, the present disclosure relates to treatment of dermal scars, wound scars, surgical scars, or cutaneous scars. Normally, cutaneous scar does not contain dermal appendages such as hair follicles and sebaceous glands, and the stem cells that typically inhabit these structures are also absent. Treatment of facial wounds with botulinum toxin A has been shown to improve cosmetic outcome in primates compared with placebo. See, Gassner et al., Plast Reconstr Surg 105:1948-1953, 2000. Accordingly, in accordance with the present disclosure, a binding domain of a botulinum toxin, as provided above, may be injected adjacent to excisional wounds on the subjects to deliver a therapeutic benefit.

In some embodiments, the present disclosure relates to treatment of hypertrophic scars or keloid scars. Botulinum toxin A has been used for the treatment of keloids by intralesional injection. See, Zhibo et al., Plast Reconstr Surg., 124(5):275-277, 2009. Accordingly, in accordance with the present disclosure, a binding domain of a botulinum toxin, as provided above, may be administered intralesionally in subjects to deliver a therapeutic benefit.

In some embodiments, the present disclosure relates to treatment of adhesions. Adhesions are fibrous bands that form between tissues and organs, often as a result of injury during surgery. They include internal scar tissue that connects tissues that are not normally connected. Botox has been evaluated in preventing postoperative intraperitoneal adhesions in laboratory animal models. See, Dokur et al., Ann Surg Treat Res., 93(1): 50-56, 2017. Accordingly, in accordance with the present disclosure, a binding domain of a botulinum toxin, as provided above, may be administered into subjects to deliver a therapeutic benefit against adhesions.

In some embodiments, the present disclosure relates to treatment of fibromas. Fibromas (or fibroids) are benign tumors that are composed of fibrous or connective tissue. They can grow in all organs, arising from mesenchyme tissue and are most prominent in eyelids. Accordingly, in accordance with the present disclosure, a binding domain of a botulinum toxin, as provided above, may be administered into subjects, either transdermally or interperitoneally to deliver a therapeutic benefit against fibromas.

In some embodiments, the present disclosure relates to treatment of liver fibrosis or cirrhosis. In some embodiments, these fibrotic diseases of the liver are treated with antifibrotics, which have been shown to reduce the likelihood that liver scarring will occur. The antifibrotic prescribed usually depends on the underlying medical condition. Examples of these treatments include, e.g., (a) for chronic liver disease: ACE inhibitors, such as benazepril, lisinopril, and Ramipril; (b) for hepatitis C virus: α-tocopherol or interferon-α; and (c) for nonalcoholic steatohepatitis: PPAR-α agonist. Accordingly, in accordance with the present disclosure, a binding domain of a botulinum toxin, as provided above, may be administered into subjects to treat liver fibrosis or cirrhosis.

In some embodiments, the present disclosure relates to treatment of pancreatitis, especially, pancreatitis caused by certain genetic disorders such as cystic fibrosis among others. Accordingly, in accordance with the present disclosure, a binding domain of a botulinum toxin, as provided above, may be administered into subjects to treat pancreatitis.

In some embodiments, the present disclosure relates to treatment of benign prostatic hyperplasia (BPH), a disease characterized by enlargement of prostate gland. Certain studies have suggested that development of BPH is a consequence of fibrosis and weakening of the muscular tissue in the prostate. Accordingly, in accordance with the present disclsoure, a binding domain of a botulinum toxin, as provided above, may be administered into subjects to treat BPH.

In some embodiments, the present disclosure relates to treatment of breast fibrosis, e.g., fibrosis after breast surgery. Breast fibrosis may be characterized by a thickening or increase in the density of breast tissue. Fibrous breast tissues include ligaments, supportive tissues (stroma), and scar tissues. Sometimes these fibrous tissues become more prominent that the fatty tissues in an area of the breast, possibly resulting in a firm or rubbery bump.

In some embodiments, the present disclosure relates to treatment of retroperitoneal fibrosis or Ormond's disease, a disease featuring the proliferation of fibrous tissue in the retroperitoneum, the compartment of the body containing the kidneys, aorta, renal tract, and various other structures. The disease is diagnosed with a CT scan and therapy may include glucocorticoids, followed by DMARDs either as steroid-sparing agents or if refractory on steroids. The SERM tamoxifen has shown to improve the condition in various small trials. Thus, in accordance with the present disclsoure, a binding domain of a botulinum toxin, as provided above, may be administered, either solely or together with one of the aforementioned drugs, to treat retroperitoneal fibrotic diseases such as fibrotic bladder, interstitial cystitis (painful bladder syndrome), pulmonary fibrosis (e.g., idiopathic pulmonary fibrosis), vascular fibrosis (e.g., atherosclerosis, stenosis, restenosis), kidney fibrosis, glomerulosclerosis, atrial fibrosis, cardiac fibrosis, endomyocardial fibrosis, arterial stiffness, arthrofibrosis, mediastinal fibrosis, myelofibrosis, retroperitoneal fibrosis, progressive massive fibrosis (e.g., lungs), chronic kidney disease (CKD), nephrogenic systemic fibrosis, using the compositions of the disclosure.

In some embodiments, the present disclosure relates to treatment of pulmonary fibrosis such as mediastinal fibrosis or progressive massive fibrosis (e.g., PMF of lungs), cystic fibrosis, non-cystic fibrosis, or asthma. PMF is characterized by the development of large conglomerate masses of dense fibrosis (usually in the upper lung zones). Subjects with pulmonary fibrosis have varying amounts of scar tissue in the mediastinum, which may cause problems for the organs located there. Treatment depends on which structures of the mediastinum are affected, the severity of the scarring and, in some cases, the cause of the condition.

In some embodiments, the disclosure relates to treatment of IPF, comprising administering, to a subject in need thereof, a therapeutically effective amount of the composition of the present disclosure. IPF is a restrictive lung disease characterized by progressive interstitial fibrosis of lung parenchyma, affecting approximately 100,000 patients in the United States (Raghu et al., Am J Respir Crit Care Med 174:810-816, 2006). This interstitial fibrosis associated with IPF leads to progressive loss of lung function, resulting in death due to respiratory failure in most patients. The median survival from the time of diagnosis is 2-3 years (Raghu et al., Am J Respir Crit Care Med 183:788-824, 2011). The prognosis is dire and often lung transplantation is the only resort (Thabut et al., Annals of Internal Medicine 151:767-774, 2009). Common symptoms of IPF include shortness of breath; persistent dry, hacking cough; fatigue; inexplicable weight loss; muscle and/or joint aches muscles; clubbing (widening and rounding of the tips of the fingers or toes).

In some embodiments, the disclosure relates to treatment of cystic fibrosis (CF), including, chronic lung disease, in a subject in need thereof, comprising administering a therapeutically effective amount of the composition of the present disclosure. In CF, bacterial colonization of the airways generally occurs within the first year or two after birth. Symptoms of cystic fibrosis include, for example, abdominal pain, chronic cough (with blood or with phlegm); gastrointestinal issues (diarrhea, fat in stool, heartburn, severe constipation, or bulky stools); respiratory problems (e.g., pulmonary hypertension, shortness of breath, sinusitis, or wheezing); developmental defects in children (e.g., delayed development, delayed puberty, or slow growth); including systemic issues such as fatigue or inability to exercise. Other common symptoms include, e.g., acute bronchitis, deformity of nails, infection, nasal polyps, pneumonia, salty sweat, or weight loss. In some embodiments, the disclosure relates to treatment of non-cystic fibrosis, comprising administering, to a subject in need thereof, a therapeutically effective amount of the composition of the present disclosure.

In some embodiments, the present disclosure relates to treatment of renal fibrosis such as chronic kidney disease (CKD) or nephrogenic systemic fibrosis (NSF) using the compositions of the disclosure. Renal ultrasonography is useful for diagnostic and prognostic purposes in CKD or NSF, e.g., in identifying whether the underlying pathologic change is due to interstitial fibrosis. For example, NSF shows a proliferation of dermal fibroblasts and dendritic cells, thickened collagen bundles, increased elastic fibers, and deposits of mucin and certain reports have described the presence of sclerotic bodies (also known as elastocollagenous balls) in skin biopsies from NSF patients. See, Scheinfeld et al., eMedicine, published: May 22, 2018.

In some embodiments, the present disclosure relates to treatment of gastrointestinal fibrotic disorders such as Crohn's disease using the compositions of the disclosure,

In some embodiments, the present disclosure relates to treatment of diseases caused by fibrosis of connective tissues, e.g., mixed connective tissue disease, ocular fibrosis, osteoarthritis, or scleroderma, using the compositions of the disclosure.

In some embodiments, the present disclosure relates to treatment of fibrotic diseases caused by systemic fibrotic conditions, e.g., systemic sclerosis (e.g., skin, lungs), athrofibrosis (e.g., knee, shoulder, other joints), using the compositions of the disclosure. Typically, to treat such systemic diseases, the compositions may be administered systemically, e.g., topically in the case of systemic sclerosis of the skin.

In some embodiments, the present disclosure relates to treatment of less common fibrotic diseases such as Peyronie's disease (fibrosis of penis), Dupuytren's contracture (bending of fingers), adhesive capsulitis (frozen shoulder), organ transplant associated fibrosis, using the compositions of the disclosure.

The disclosure further relates to a composition comprising a binding domain of a botulinum toxin, as provided above, for use in treatment of a disease selected from the group consisting of extracellular matrix (ECM) disease, connective tissue disease, endothelial disease or fibrotic disease; preferably, fibrotic disease of the ECM, connective tissue, or endothelium.

In some embodiments, the disclosure relates to a composition comprising a binding domain of a botulinum toxin, as provided above, for use in treatment of an extracellular matrix (ECM) disease. In some embodiments, the disclosure relates to a composition comprising a binding domain of a botulinum toxin, as provided above, for use in treatment of a connective tissue disease. In some embodiments, the disclosure relates to a composition comprising a binding domain of a botulinum toxin, as provided above, for use in treatment of an endothelial disease. In some embodiments, the disclosure relates to a composition comprising a binding domain of a botulinum toxin, as provided above, for use in treatment of a fibrotic disease.

In some embodiments, the disclosure relates to a composition for use in treatment of a fibrotic disease selected from dermal scars, wound scars, surgical scars, postoperative intraperitoneal adhesions, fibroma, liver fibrosis, cirrhosis, pancreatitis, benign prostatic hyperplasia, breast fibrosis, fibrotic bladder, interstitial cystitis, pulmonary fibrosis (e.g., idiopathic pulmonary fibrosis), vascular fibrosis (e.g., atherosclerosis, stenosis, restenosis), kidney fibrosis, glomerulosclerosis, atrial fibrosis, cardiac fibrosis, endomyocardial fibrosis, glial scar, arterial stiffness, arthrofibrosis, mediastinal fibrosis, myelofibrosis, retroperitoneal fibrosis, progressive massive fibrosis (e.g., lungs), chronic kidney disease (CKD), nephrogenic systemic fibrosis, Crohn's disease, hypertrophic scarring, keloid, scleroderma, systemic sclerosis (e.g., skin, lungs), athrofibrosis (e.g., knee, shoulder, other joints), Peyronie's disease, Dupuytren's contracture, adhesive capsulitis, organ transplant associated fibrosis, ischemia associated fibrosis, cystic fibrosis, non-cystic fibrosis, mixed connective tissue disease, ocular fibrosis, osteoarthritis, scleroderma, or asthma, preferably, treatment of a fibrotic disease selected from fibrotic breast disease; keloid scar; liver fibrosis; fibrotic bladder; hyperplasia of fibrous tissue, e.g., benign prostatic hyperplasia; pancreatitis; adhesion, e.g., intra-abdominal adhesion; scarring, e.g., hypertrophic scars or a combination thereof.

The disclosure further relates to use of a composition comprising a binding domain of a botulinum toxin, as provided above, for the manufacture of a medicament for treating a disease selected from the group consisting of extracellular matrix (ECM) disease, connective tissue disease, endothelial disease or fibrotic disease; preferably, for treating a fibrotic disease of the ECM, connective tissue, or endothelium.

In some embodiments, the disclosure relates to use of a composition comprising a binding domain of a botulinum toxin, as provided above, for the manufacture of a medicament for treating an extracellular matrix (ECM) disease. In some embodiments, the disclosure relates to use of a composition comprising a binding domain of a botulinum toxin, as provided above, for the manufacture of a medicament for treating a connective tissue disease. In some embodiments, the disclosure relates to use of a composition comprising a binding domain of a botulinum toxin, as provided above, for the manufacture of a medicament for treating an endothelial disease. In some embodiments, the disclosure relates to use of a composition comprising a binding domain of a botulinum toxin, as provided above, for the manufacture of a medicament for treating a fibrotic disease.

In some embodiments, the disclosure relates to use of a composition comprising a binding domain of a botulinum toxin, as provided above, for the manufacture of a medicament for treating a fibrotic disease selected from dermal scars, wound scars, surgical scars, postoperative intraperitoneal adhesions, fibroma, liver fibrosis, cirrhosis, pancreatitis, benign prostatic hyperplasia, breast fibrosis, fibrotic bladder, interstitial cystitis, pulmonary fibrosis (e.g., idiopathic pulmonary fibrosis), vascular fibrosis (e.g., atherosclerosis, stenosis, restenosis), kidney fibrosis, glomerulosclerosis, atrial fibrosis, cardiac fibrosis, endomyocardial fibrosis, glial scar, arterial stiffness, arthrofibrosis, mediastinal fibrosis, myelofibrosis, retroperitoneal fibrosis, progressive massive fibrosis (e.g., lungs), chronic kidney disease (CKD), nephrogenic systemic fibrosis, Crohn's disease, hypertrophic scarring, keloid, scleroderma, systemic sclerosis (e.g., skin, lungs), athrofibrosis (e.g., knee, shoulder, other joints), Peyronie's disease, Dupuytren's contracture, adhesive capsulitis, organ transplant associated fibrosis, ischemia associated fibrosis, cystic fibrosis, non-cystic fibrosis, mixed connective tissue disease, ocular fibrosis, osteoarthritis, scleroderma, or asthma, preferably, treatment of a fibrotic disease selected from fibrotic breast disease; keloid scar; liver fibrosis; fibrotic bladder; hyperplasia of fibrous tissue, e.g., benign prostatic hyperplasia; pancreatitis; adhesion, e.g., intra-abdominal adhesion; scarring, e.g., hypertrophic scars or a combination thereof.

It is further contemplated that the polypeptides or compositions of the disclosure may be used in combination with each other (e.g., BoNT/A H_(c) fragment of SEQ ID NO: 1 and mutant BoNT/A H_(c) fragment of SEQ ID NO: 25) or with other antifibrotic agents to modulate the activity of the composition. Combinations of the peptide or compounds with other agents may be useful to allow antifibrotic to be used at lower doses due to toxicity concerns, to enhance the activity of antifibrotic agents whose efficacy has been reduced or to effectuate a synergism between the components such that the combination is more effective than the sum of the efficacy of either component independently. Antifibrotic agents which may be combined with an antimicrobial peptide in combination therapy include, e.g., pirfenidone and nintedanib, which have been approved for treatment of IPF.

In practicing the aforementioned therapeutic applications, it is advantageous to use compositions containing Clostridial toxins or fragments or variants thereof which do not produce paralysis of a facial muscle.

EXAMPLES

The structures, materials, compositions, and methods described herein are intended to be representative examples of the disclosure, and it will be understood that the scope of the disclosure is not limited by the scope of the examples. Those skilled in the art will recognize that the disclosure may be practiced with variations on the disclosed structures, materials, compositions and methods, and such variations are regarded as within the ambit of the disclosure.

Example 1 Effect of an Exemplary Polypeptide of SEQ ID NO: 19 on Cellular Gene Expression in Normal Human Primary Fibroblasts

Treatment of normal human primary fibroblasts with 10 nM, 100 nM or 1 μM of a polypeptide having an amino acid sequence substantially identical to the amino acid sequence of the binding domain of BoNT/A (H_(C)/A) for 1 day (24 hours) resulted in significant dose-dependent changes in expression of 16 fibroblast genes based on qPCR. Briefly, Human Dermal Fibroblast, adult (HDFa) (ThermoFisher Scientific, Cat. No. C0135C) were cultured in MEM medium containing 2% FBS for 2 weeks before treatment with 10 nM, 100 nM or 1 μM of a polypeptide of SEQ ID NO: 19 for 1 day (24 hours). Expression of genes known to be expressed in fibroblasts and involved with ECM organization, inflammation, or epidermal self-renewal (keratinocyte stem cell factor) were evaluated. cDNA was generated by reverse transcription using SUPERSCRIPT VILO cDNA Synthesis Kit (Thermo Scientific #11754050) and further diluted in TAQMAN Fast Advanced Master Mix (Thermo Scientific #4444557) before transfer to designated wells in TAQMAN Fast plates (Thermo Scientific #4413259). Real-time qPCR was performed using the Applied Biosystems 7500 Fast Real-Time PCR system (Thermo Fisher Scientific). Changes in gene expression were calculated as fold change over the untreated control at each time point (ΔΔCT=ΔCT (Gene (test)−GAPDH (test))−ΔCT (Gene (untreated control)−GAPDH (untreated control)); Fold Change=2(−ΔΔCT)). Fold changes greater than 2 or less than 0.5 (p-value <0.05) were considered relevant significant changes. Exemplary genes showing a dose-dependent change are shown in FIG. 1, which is a bar graph showing the fold-change in expression of the indicated genes in normal human primary fibroblast cells after treatment with 10 nM (solid bar), 100 nM (unfilled bar) or 1 μM (hatched bar) of a polypeptide having an amino acid sequence substantially identical to the amino acid sequence of the binding domain of BoNT/A (H_(C)/A) for 24 hours, where the fold-change is expressed relative to untreated control cells. The results show that genes known to be involved in extracellular matrix breakdown and re-modeling, including MMP1, MMP3, TIMP1, were up-regulated; while genes associated with fibrosis, including S100 calcium-binding protein A4 (S100A4) and inducer of alpha-smooth muscle actin, and alpha-smooth muscle actin (α-SMA/ACTA2) were down-regulated. S100A4, a known marker of fibrosis, and α-SMA/ACTA2 are associated with fibroblast contractility and myofibroblast formation. Since fibroblast contractility and myofibroblast formation as well as excess production of S100A4, ACTA2 and other ECM components play a role in scar formation and fibrosis, these results suggest that the polypeptide of SEQ ID NO: 19 would have anti-scarring and anti-fibrotic effects. The results suggest that polypeptides corresponding substantially to the binding domain of BoNT/A (H_(C)/A) could have an effect on ECM structure and fibrosis in human patients, e.g. fibrous tissues and organs, and scars, resulting in for example inhibition or reversal of tissue and organ fibrosis and visual scar reduction.

Example 2 Effect of an Exemplary Polypeptide of SEQ ID NO: 21 on Cellular Gene Expression in Keloid Scar Derived Human Primary Fibroblasts

Treatment of keloid scar human primary fibroblasts with a polypeptide having an amino acid sequence substantially identical to the amino acid sequence of the N-terminal half of the binding domain of BoNT/A (H_(CN)/A) (SEQ ID NO: 21) resulted in significant dose-dependent changes in expression of six (6) fibroblast genes based on qPCR. Briefly, human Dermal Fibroblasts, (KF116R) (cellResearchCorp Pte Ltd) were cultured in MEM medium containing 2% FBS for 2 weeks before treatment with 1 nM, 10 nM, 100 nM or 1 μM of a polypeptide of SEQ ID NO: 21 for 1 day (24 hours). Expression of six (6) fibroblast-related genes, known to be involved with ECM organization and re-modeling (MMP1, MMP3, and TIMP1), and epidermal differentiation and self-renewal (FGFR1, FGF7, and TP63) was evaluated. cDNA was generated by reverse transcription using SUPERSCRIPT VILO cDNA Synthesis Kit (ThermoScientific #11754050) and further diluted in TAQMAN Fast Advanced Master Mix (ThermoScientific #4444557) before transfer to designated wells in TAQMAN Fast plates (ThermoScientific #4413259). Real-time qPCR was performed using the Applied Biosystems 7500 Fast Real-Time PCR system (Thermo Fisher Scientific). Changes in gene expression were calculated as fold change over the untreated control at each time point (ΔΔCT=ACT (Gene (test)−GAPDH (test))−ΔCT (Gene (untreated control)−GAPDH (untreated control)); Fold Change=2(−ΔΔCT)). Fold changes greater than 2 or less than 0.5 (p-value <0.05) are considered significant changes. Exemplary genes showing a dose-dependent change are shown in FIG. 2, which is a bar graph showing the fold change in expression of the indicated genes in keloid scar human primary fibroblasts after treatment with 1 nM (horizontal line fill), 10 nM (unfilled bar), 100 nM (hatched bar) or 1 (solid bar) of a polypeptide having an amino acid sequence substantially identical to the N-terminal half of the binding domain of BoNT/A (H_(CN)/A) (SEQ ID NO: 21). The results show that treatment of the cells with the polypeptide of SEQ ID NO: 11 changed expression of proteins involved with creating, maintaining and repairing the dermis Extra Cellular Matrix (ECM). Since excess production of ECM components plays a role in scar formation and fibrosis, these results suggest that the polypeptide of SEQ ID NO: 21 would have anti-scarring and anti-fibrotic effects. The results suggest that polypeptides corresponding substantially to the binding domain of BoNT/A (H_(C)/A) or the N-terminal half of the binding domain (H_(CN)/A) could affect fibrosis in human patients, e.g. fibrous tissues and organs, and scars, resulting in for example inhibition or reversal of tissue and organ fibrosis and visual scar reduction.

Example 3 Effect of an Exemplary Polypeptide of SEQ ID NO: 19 on Procollagen Secretion from Keloid Scar Derived Human Primary Fibroblasts

Treatment of keloid scar human primary fibroblasts with 1 μM of a polypeptide having an amino acid sequence substantially identical to the binding domain of BoNT/A (H_(C)/A) (SEQ ID NO: 19) resulted in significant reduction, ˜30% in Procollagen Type I C-peptide (PIP) secretion into media with 10% FBS from keloid scar human primary fibroblasts. The reduction was similar to the reduction observed after treatment with either 0.1 μM of an antibody to TGF-beta, ˜20%, or 1 μM of an antibody to CTGF, ˜30%. Briefly, human dermal keloid fibroblasts (KF116R) were regularly cultured in MEM medium containing 2% FBS. For the experiment, cells were either kept in 2% FBS medium or switched to medium containing 10% FBS, with or without either 1 μM of a polypeptide of SEQ ID NO:19, 0.1 μM of anti-TGF-β mAb, or 1 μM of Anti-CTGF mAb treatment. After 24 hours, media was collected and Procollagen Type I C-peptide (PIP) level was measured using Procollagen Type I C-Peptide (PIP) EIA Kit (Takara BIO INC. Cat. #MK101). Data are presented in FIG. 3, which is a bar graph showing the amount (ng/mL) of PIP secreted into media from keloid scar human primary fibroblasts after culturing in 2% FBS or culturing in 10% FBS with or without treatment with either 1 μM of a polypeptide having an amino acid sequence substantially identical to the binding domain of BoNT/A (H_(C)/A) (SEQ ID NO: 19); 0.1 μM of an antibody to TGF-beta, or 1 μM of an antibody to CTGF, two known anti-fibrotic agents. The results show that the polypeptide of SEQ ID NO:19 was as effective as the two known anti-fibrotic agents in reducing secretion of procollagen peptides, the source of mature collagen fibers, from primary dermal scar derived fibroblasts. Since excess collagen production plays a role in scar formation and fibrosis, these results suggest that the polypeptide of SEQ ID NO: 19 would have anti-scarring and anti-fibrotic effects. The results suggest that polypeptides corresponding substantially to the binding domain of BoNT/A (H_(C)/A) could affect fibrosis by reducing for example collagen production, in human patients, resulting in inhibition or reversal of tissue and organ fibrosis and visual scar reduction.

Example 4 Effect of an Exemplary Polypeptide of SEQ ID NO: 19 on Keloid Scar Derived Human Primary Fibroblast Contractility

Pre-treatment of keloid scar human primary fibroblasts with 1 μM of a polypeptide having an amino acid sequence substantially identical to the binding domain of BoNT/A (H_(C)/A) (SEQ ID NO: 19) resulted in significant reduction of fibrin hydrogel contractility, a measure for fibroblast contractility and fibroblast-myofibroblast transition, ˜50% after 1 day (24 hours). Briefly, Human Dermal Keloid Fibroblasts (KF116R) were regularly cultured in MEM medium containing 2% FBS. For the experiment, cells were either kept in 2% FBS medium or switched to medium containing 10% FBS, with or without 1 μM of a polypeptide of SEQ ID NO.: 19. After 3-days the cells were trypsinized and 1×106 cells/hydrogel were embedded into fibrin hydrogel, which was a mixture of 1 mg/mL fibrinogen and 2.5 U/mL thrombin containing MEM medium, 2% or 10% dependent on the treatment paradigm. Photographs were taken after 1 day (24 hours), at a fixed distance, and the area (cm²) of the fibrin hydrogel was calculated using Image J software. Data are presented in FIG. 4, which is a bar graph showing the size (cm²) of fibrin hydrogels with keloid scar human primary fibroblasts one day after embedding and culturing in 2% FBS media or 10% FBS media with or without 3 days pre-treatment with 1 μM of a polypeptide having an amino acid sequence substantially identical to the binding domain of BoNT/A (H_(C)/A) (SEQ ID NO: 19). The results show that treatment with 1 μM of the polypeptide of SEQ ID NO: 19 reduced fibroblast contractility and fibroblast-myofibroblast transition of keloid scar human primary fibroblasts. Fibroblast contractility is associated with fibroblast to myofibroblast transition and scar formation, for example as the fibroblasts contract to close a wound or transform an existing scar. The results suggest that polypeptides corresponding substantially to the binding domain of BoNT/A (H_(C)/A) are effective in reducing fibroblast contractility and fibroblast-myofibroblast transition, and therefore could affect fibrosis, e.g. fibrous tissues and organs, and scars in human patients, resulting in for example less scarring during wound healing and/or reversal of existing tissue and organ fibrosis and scarring.

Example 5 Effect of Exemplary Polypeptides of SEQ ID NOs: 19 and 21 on Fibrotic Breast Cancer Cells

Treatment of fibrotic epithelial human breast adenocarcinoma cancer (MDA-MB-231) cells, where fibrosis is induced by hypoxia, with 20 pM, 0.1 μM, or 1 μM of either a polypeptide having an amino acid sequence substantially identical to the binding domain of BoNT/A (HC/A) (SEQ ID NO: 19) or a polypeptide having an amino acid sequence substantially identical to the N-terminal half of the binding domain of BoNT/A (H_(CN)/A) (SEQ ID NO: 21) resulted in dose-dependent significant reduction in secretion of fibronectin (a high-molecular weight (˜440 kDa) glycoprotein of the extracellular matrix that binds to integrins, collagen, fibrin, and heparan sulfate proteoglycans (e.g. syndecans), collagen IV (a type of collagen found primarily in the basal lamina, a layer of extracellular matrix secreted by the epithelial cells), and thrombospondin-1 (an adhesive glycoprotein that binds to fibronectin and collagen and mediates cell-to-cell and cell-to-matrix interactions) into the media, and increased secretion of Matrix metalloproteinase-1 (MMP1), an enzyme that functions to break down extracellular matrix, including collagen, and TIMP metallopeptidase inhibitor 1 (TIMP1), an inhibitory regulator of matrix metalloproteinases, based on magnetic bead immunosorbent analysis. Treatment with 1 μM of a polypeptide of SEQ ID NO: 19 reduced secretion of both fibronectin, collagen IV, and thrombospondin-1, back to, or close to, the levels for normoxic cells; and treatment with a polypeptide of SEQ ID NO: 21 reduced secretion of fibronectin and thrombospondin-1, below the levels for normoxic cells, and secretion of collagen IV, back to the level for normoxic cells. In comparison, 333 nM of an antibody to CTGF (FG-3019), a known anti-fibrotic agent, did not affect secretion of thrombospondin-1, showed less inhibitory effect on fibronectin secretion, and similar inhibitory effect on collagen IV secretion. Briefly, MDA-MB-231 cells were acquired from ATCC (Rockville, Md.) and cultured at 37° C. and 5% CO2 in DMEM media supplemented with 10% FBS and 1% antibiotics. Cells were seeded at 2×105 cells/ml in 12-well plates. The next day, complete medium was removed, cells were washed with 1×PBS and kept in serum-free media with or without a polypeptide of SEQ ID NO.: 19 and a polypeptide of SEQ ID NO.: 21 at 3 different concentrations: 20 pM, 0.1 and 1 uM and the anti-CTGF antibody, FG-3019 at 333 nM final concentration (n=2, 2-wells per condition). Untreated cells were incubated either; a) under normoxia (˜21% O2) in a standard CO2 incubator or b) under hypoxia (1% O2) in a humidified triple gas model (Binder) incubator. All treated cells were kept under low oxygen conditions for 120 hours (h) and cell culture supernatants were collected and analyzed using magnetic bead immunosorbent assays (EMD Millipore, Md.). Levels of pro-fibrotic markers secreted in the cell culture media was determined using an ELISA-like technology: Magnetic beads (Luminex) consisting of antibody-conjugated beads against specific proteins (custom panel), including fibronectin, collagen IV, thrombospondin-1, MMP1, and TIMP1. The media concentration of each analyte was determined using a standard solution containing all the proteins in the panel. Data are presented in FIG. 5, which is bar graphs showing the amount (ng/mL) of fibronectin, collagen IV, thrombospondin-1, MMP1, and TIMP1 secreted into the media from fibrotic MDA-231 cells, where fibrosis is induced by hypoxia, after treatment with 20 pM, 0.1 μM, or 1 μM of either a polypeptide having an amino acid sequence substantially identical to the binding domain of BoNT/A (H_(C)/A), a polypeptide having an amino acid sequence substantially identical to the N-terminal half of the binding domain of BoNT/A (H_(CN)/A), or 333 nM of an antibody to CTGF (FG-3019). The results show that treatment with a polypeptide of SEQ ID NO: 19 or a polypeptide of SEQ ID NO: 21 was effective in reducing secretion of fibronectin, collagen IV, thrombospondin-1, and increasing secretion of MMP1 and TIMP1, from fibrotic MDA-MB-231 cells, and that the effect was comparable or stronger relative to the effect of the known anti-fibrotic agent FG-3019. The results suggest that polypeptides corresponding substantially to the binding domain of BoNT/A (H_(C)/A) or the N-terminal half of the binding domain are effective in reducing hypoxia induced fibrosis.

Example 6 Effect of an Exemplary Polypeptide of SEQ ID NO: 19 on Secretion of Fibronectin from Keloid Scar Derived Fibroblasts

Treatment of fibrotic human keloid scar fibroblast (KF116R) cells with 20 pM, 200 pM, 20 nM, or 200 nM of a polypeptide having an amino acid sequence of SEQ ID NO: 19 for 1, 4, 7, or 9 days, resulted in a significant time and dose-dependent reduction in secretion of fibronectin (a high-molecular weight (˜440 kDa) glycoprotein of the extracellular matrix that binds to integrins, collagen, fibrin, and heparan sulfate proteoglycans (e.g. syndecans), based on fibronectin enzyme immunoassay (EIA). Briefly, KF116R cells were acquired from CellResearchCrop (Singapore) and cultured at 37° C. and 5% CO2 in MEM media supplemented with 2% FBS and 1% antibiotics. The cells were seeded at 2×105 cells/mL in a 24-well plate for one day before treatment with either 20 pM, 200 pM, 20 nM, or 200 nM of a polypeptide of SEQ ID NO: 19. For the 7 and 9 days treatments, media with treatment was replaced on day 5. For measurement of fibronectin, the media was collected at 1, 4, 7, and 9 days after the first treatment, and the amount of fibronectin secreted into the media (ng/mL) was measured using Takara Fibronectin EIA kit (Clontech, Cat#MK115). Data are presented in FIG. 6, which is a bar graph showing the normalized amount of fibronectin, normalized to the media only control for each timepoint, secreted into the media from human keloid scar fibroblast (KF116R) cells, after treatment with 20 pM, 200 pM, 20 nM, or 200 nM of a polypeptide having an amino acid sequence of SEQ ID NO: 19. The results show that treatment with the polypeptide of SEQ ID NO: 19 was effective in reducing secretion of fibronectin from human keloid scar fibroblast (KF116R) cells. Since formation of scars and fibrosis is associated with excess production of fibronectin and other ECM components, the results suggest that polypeptides corresponding substantially to the binding domain of BoNT/A (H_(C)/A) have anti-scarring and anti-fibrotic effects.

Example 7 Effect of an Exemplary Polypeptide of SEQ ID NO: 19 on Protein Secretion from Normal Human Primary Fibroblasts

Treatment of normal human primary fibroblasts (HDFa) with 20 pM of a polypeptide having an amino acid sequence of SEQ ID NO: 19 for 6 days resulted in a significant reduction in secretion of collagens (Collagen alpha-1(I) chain, 57%, Collagen alpha-1(III) chain, 87%, Collagen alpha-2(I) chain, 47%) and lactotransferrin (53%) based on Mass Spectrometry (MS) protein analysis of collected media. Briefly, Human Dermal Fibroblast, adult (HDFa) (ThermoFisher Scientific, Cat. No. C0135C) were cultured in MEM medium containing 2% FBS for 2 weeks before treatment with 20 pM of a polypeptide of SEQ ID NO: 19 for 6 days, followed by incubation in media only for 3 days (n=3). The media was collected, and proteins were identified and quantified by Mass Spectrometry (MS) protein analysis. Data are presented in FIG. 7, which is a bar graph showing the identified proteins and the average amount of each protein in the media collected from untreated and treated primary human fibroblasts. The results show that treatment with the polypeptide of SEQ ID NO: 19 was effective in reducing secretion of collagens and lactotransferrin, the latter has been shown to increase fibroblast contractility from normal human primary fibroblasts. Since formation of scars and fibrosis is associated with excess production of collagen and fibroblast contractility, the results suggest that polypeptides corresponding substantially to the binding domain of BoNT/A (H_(C)/A) have anti-scarring and anti-fibrotic effects.

Example 8 Effect of Exemplary Polypeptides of SEQ ID NOs: 20 and 21 on Cellular Gene Expression in Keloid Scar Derived Human Fibroblasts

Treatment of keloid scar human primary fibroblasts with 100 nM or 1 μM of a polypeptide having an amino acid sequence substantially identical to the binding domain of BoNT/DC (H_(C)/DC) (SEQ ID NO: 20) or 1 μM of a polypeptide having an amino acid sequence substantially identical to the N-terminal half of the binding domain of BoNT/A (H_(CN)/A) (SEQ ID NO: 21) resulted in significant changes in expression of fibroblast-related genes, based on qPCR. Briefly, Human Dermal Keloid Fibroblasts (KF116R) were cultured in MEM medium containing 2% FBS for 2 weeks before treatment with 100 nM or 1 μM of a polypeptide of SEQ ID NO.: 20 or 1 μM of a polypeptide of SEQ ID NO.: 21 for 1 day (24 hours). Expression of fibroblast-related genes, known to be involved with ECM organization (COL1A1, COL1A2, COL3A1, COL4A1, COL6A3, COL7A1, ELN, FBN1, and FN1) and re-modeling (MMP1, MMP2, MMP3, and TIMP1) and fibrosis and fibroblast-myofibroblast transition (TGFB1, TGFB3, TGFBR1, TGFBR2, CTGF, S100A4, VIM, ACTA2, CD34, and SERPINE1) was evaluated. cDNA was generated by reverse transcription using SUPERSCRIPT VILO cDNA Synthesis Kit (ThermoScientific #11754050) and further diluted in TAQMAN Fast Advanced Master Mix (ThermoScientific #4444557) before transfer to designated wells in TAQMAN Fast plates (ThermoScientific #4413259). Real-time qPCR was performed using the Applied Biosystems 7500 Fast Real-Time PCR system (Thermo Fisher Scientific). Changes in gene expression were calculated as fold change over the untreated control at each time point (ΔΔCT=ΔCT (Gene (test)−GAPDH (test))−ΔCT (Gene (untreated control)−GAPDH (untreated control)); Fold Change=2(−ΔΔCT)). Fold changes greater than 2 or less than 0.5 (p-value <0.05) are considered significant changes. Exemplary genes (23) are shown in FIG. 8, which is a bar graph showing the fold change in expression of the indicated genes in keloid scar human primary fibroblasts after treatment with 100 nM (solid bar) or 1 μM (hatched bar) of the polypeptide having an amino acid sequence of SEQ ID NO: 20 or 1 μM (unfilled bar) of a polypeptide having an amino acid sequence of SEQ ID NO: 21. The results show that treatment with the polypeptides of SEQ ID NO: 20 and SEQ ID NO: 21 affected expression of genes known to be involved ECM organization and re-modeling and fibrosis and fibroblast-myofibroblast transition; and the effect induced by the polypeptide of SEQ ID NO: 20 appeared more significant than the effect induced by the polypeptide of SEQ ID NO: 21. The results suggest that polypeptides corresponding substantially to either the binding domain of BoNT/DC (H_(C)/DC) or the N-terminal half of the binding domain (H_(CN)/A) have anti-scarring and anti-fibrotic effects. The observation that polypeptides corresponding substantially to the binding domains of two different BoNT serotypes affect gene expression in keloid scar derived human fibroblasts suggests that other additional BoNT serotypes may have the same anti-scarring and anti-fibrotic effects.

Using a sequence alignment software tool, pairwise sequence alignment was performed between different BoNT serotypes,; wherein the amino acid sequence of the binding domain of BoNT/A1 (H_(C)/A) (SEQ ID NO: 1) (GENBANK #AF488749) was aligned with the amino acid sequence of the binding domain from the following BoNT proteins: BoNT/B1 (GENBANK #BAE48264): BoNT/C1 (GENBANK #P18640); BoNT/D (GENBANK #P19321); BoNT/DC (GENBANK #EF378947); BoNT/E (GENBANK #AFV91344); BoNT/F (GENBANK #ABS41202); and BoNT/G (GENBANK #X74162). The results, which are shown in Table 2, revealed that the percent identity and homology at the amino acid levels between BoNT/A1 and other BoNT serotypes, e.g., B1, C1, DC, E, F, and G, is similar to the percent identity and homology between BoNT/A1 and BoNT/DC. Specifically, according to the BLAST alignment H_(C)/A and H_(C)/DC are 33% identical and 54% similar (consensus) at the amino acid residue level, which is similar to all the other serotypes (31-51% identical and 49-67% similar (consensus)). As shown in Example 5, the binding domain of BoNT/DC (H_(C)/DC) was as effective as binding domain of BoNT/A in affecting fibroblast. Thus, the BLAST alignment and the results obtained from Example 5 suggest that other BoNT serotypes, in addition to BoNT/A and BoNT/DC, can affect human skin.

TABLE 2 Pairwise multiple sequence alignment between Hc proteins derived from various BoNT serotypes using BLAST. Comparison Variant (Strain) Tool Identity (%) Consensus (%) Homology (%)* Gaps (%) Score** H_(C)/A (Hall A) H_(CN)/A (Hall A) BLAST 218/218 (100%) 218/218 (100%) 51.78 0/218 (0%) 435 bits(1118) H_(CN)/A 40-mer (Hall A) 40/40 (100%) 40/40 (100%) 9.50 0/40 (0%) 82.0 bits(202) H_(C)/B (

) 178/440 (40%) 257/440 (58%)

1.05 37/440 (8%) 2

3 bits(723) H_(C)/C (Stockholm) 136/442 (31%) 217/442 (49%) 51.34 49/442 (11%) 166 bits(420) H_(C)/D (D-1873) 137/434 (32%) 226/434 (52%) 53.68 40/434 (9%) 182 bits(462) H_(C)/DC (VP

5995) 117/350 (33%) 189/350 (54%) 44.89 30/350 (9%) 166 bits(421) H_(C)/C (CDC41648) 202/424 (48%) 272/424 (64%) 64.61 26/424 (6%) 330 bits(345) H_(C)/F (Langeland) 214/422 (51%) 286/422 (67%)

7.93 19/422 (4%) 395 bits(1014) H_(C)/G (11330) 176/436 (40%) 247/436 (56%) 58.67 26/436 (6%) 273 bits(638) *The percent homology is defined as the percent of either identical or similar residues (Consensus) within a protein sequence relative to a reference protein sequence divided by the length of the reference sequence **The ( ) score is the raw score directly computed using the matrix of residue substitution. The bit score is a normalized score which considered the sequence length and gap size. For example, 283 bits means to find a better alignment than the one you found, you have to search 2{circumflex over ( )}283 amino acids space.

indicates data missing or illegible when filed

As shown in Table 2, the level of alignment between different BoNT serotypes is very high, as indicated by the score. The score is provided either in the form of a raw score or a bit score, wherein the raw score is directly computed by the tool using the matrix of residue substitution and the bit score is a normalized score, which considers the sequence length and gap size. As is understood in bioinformatics, a score of 283 bits means to find a better alignment than what is presented, the search would have to encompass an amino acids space of 2²⁸³ (or 2×10⁸⁵) units. Thus, the higher the bit score, the more highly significant the match.

Example 9 Use of an Exemplary Polypeptide Provided in Accordance with Aspects of the Present Disclosure to Treat Keloid Scar

A 20-year African female with photo type VI skin presents with a 2-year old keloid scar her left ear that developed after an ear piercing. She receives treatment with a H_(C)/A polypeptide once a month for a total period of three months.

The H_(C)/A polypeptide powder is dissolved in saline (4.5 ng vacuum-dried powder was reconstitution in 0.25 mL of sterile 0.9% saline) to constitute a solution at 1.8 ng/0.1 mL and injects into the body of her scar using a 9-gauge needle (0.3 mm×0.8 mm) until slight blanching is visible. The dose is 0.9 ng/cm³ of the lesion.

Evaluation is Conducted at Baseline and at 6 Months' Post-Treatment.

Compared to baseline, at 6 months' post-treatment, her scar shows good to excellent improvement based on the physician's global assessment and subject satisfaction score, with significant improvement in erythema, itching and pliability. This improvement is consistent with experimental data described in Examples 1, 3, 5, 6 and 7, wherein fibroblasts treated with a polypeptide corresponding to the binding domain of BoNT/A (H_(C)/A) were shown to have reduced expression of genes associated with myofibroblast formation, including S100A4 and ACTA2, and modulated expression and secretion of extracellular matrix (ECM) proteins and proteinases, including fibronectin, collagen and MMP1, that function to properly maintain, renew and repair ECM dermal tissue structures.

Example 10 Use of an Exemplary Polypeptide Provided in Accordance with Aspects of the Present Disclosure to Treat Liver Fibrosis

A 55-year male with a history of alcohol abuse presents with complains of fatigue and loss of appetite. From blood testing it is determined that he has elevated liver enzymes and after confirming by liver biopsy, he is diagnosed with liver fibrosis, 2nd stage liver disease. His hepatic volume (liver size) is determined by magnetic resonance imaging (MM) to be approximately 1000 cm3. He receives treatment with a polypeptide having an amino acid sequence which is substantially identical to the binding domain of BoNT/A (H_(C)/A) once a month for a total period of three months.

The (H_(C)/A) polypeptide powder is dissolved in saline, 4.5 ng vacuum-dried powder is dissolved in 0.25 mL of sterile 0.9% saline to constitute a solution at 1.8 ng/0.1 mL, and injected percutaneously using ultrasound visual guidance into the liver. The dose is 0.9 ng/cm3 of the affected region of the liver. A total of 90 ng is injected with 50 μL (0.9 ng) at each (100) injection sites.

Evaluation is Conducted at Baseline and at 6 Months' Post-Treatment.

Compared to baseline, at 6 months' post-treatment, a significant reduction in liver enzymes and liver size is measured and the patient reports improvement in energy level and appetite. This improvement is consistent with experimental data described in Examples 1, 3, 5, 6 and 7, wherein fibroblasts treated with a polypeptide corresponding to the binding domain of BoNT/A (H_(C)/A) were shown to have reduced expression of genes associated with myofibroblast formation, including S100A4 and ACTA2, and modulated expression and secretion of extracellular matrix (ECM) proteins and proteinases, including fibronectin, collagen and MMP1, that function to properly maintain, renew and repair ECM tissue structures.

Example 11 Use of an Exemplary Polypeptide Provided in Accordance with Aspects of the Present Disclosure to Treat Fibrotic Bladder

A 55-year male patient presents with symptoms of fibrotic bladder, including a chronic need to urinate, up to fifty times a day. He is referred to the urology clinic for treatment. The referred urologist is recommending that H_(C)/A peptide be used to treat his condition. A solution containing a 1 μM concentration of H_(C)/A is injected into 30 sites per approved injection paradigm (15 sites in the bladder base (including 2 in the trigone) and 15 sites at and below the bladder midline in the postero-lateral wall). At checkup, two weeks after the second treatment, the patient reports a significant improvement in equality of life, with a 50% reduction in need to urinate. This improvement is consistent with experimental data described in Examples 1, 3, 5, 6 and 7, wherein fibroblasts treated with a polypeptide corresponding to the binding domain of BoNT/A (H_(C)/A) were shown to have reduced expression of genes associated with myofibroblast formation, including S100A4 and ACTA2, and modulated expression and secretion of extracellular matrix (ECM) proteins and proteinases, including fibronectin, collagen and MMP1, that function to properly maintain, renew and repair ECM tissue structures.

Example 12 Use of an Exemplary Polypeptide Provided in Accordance with Aspects of the Present Disclosure to Treat Symptoms of Benign Prostatic Hyperplasia

A 2-stage, multicenter, double-blind, randomized phase II clinical trial with two doses of H_(C)/A peptide to treat the lower urinary tract symptoms of benign prostatic hyperplasia is conducted.

Men 50 years old or older with clinically diagnosed benign prostatic hyperplasia, American Urological Association symptom index 8 or greater, maximum urinary flow rate less than 15 ml per second, voided volume 125 ml or greater, and post-void residual 350 ml or less are randomized to prostatic transrectal injection of 2.5 or 5 ng of H_(C)/A peptide. The primary outcome is at least 30% improvement from baseline to 3 months in American Urological Association symptom index and/or maximum urinary flow rate and safety. The men are followed for 12 months

A total of 134 men are randomized and treated (68 with 2.5 ng, 66 with 5 ng), with 131 assessed at 3 months and 108 assessed at 12 months. Each dose meets the 3-month primary outcome criteria. In the 2.5 ng arm the mean baseline American Urological Association symptom index of 18.8 decreases by 7.1 and 6.9 at 3 and 12 months, respectively. In the 5 ng arm the baseline of 19.5 decreases by 8.9 and 7.1, respectively. In the 2.5 unit arm the mean baseline maximum urinary flow rate of 10.0 ml per second increases by 2.5 and 2.2, respectively, and in the 5 ng arm the baseline of 9.6 increases by 2.6 and 2.3, respectively.

The intraprostatic injection of 2.5 ng or 5 ng units of H_(C)/A peptide passes predetermined criteria for treatment efficacy and safety, and a randomized trial with either dose is warranted. The 2.5 ng unit dose may be preferable due to similar efficacy. This improvement is consistent with experimental data described in Examples 1, 3, 5, 6 and 7, wherein fibroblasts treated with a polypeptide corresponding to the binding domain of BoNT/A (H_(C)/A) were shown to have reduced expression of genes associated with myofibroblast formation, including S100A4 and ACTA2, and modulated expression and secretion of extracellular matrix (ECM) proteins and proteinases, including fibronectin, collagen and MMP1, that function to properly maintain, renew and repair ECM tissue structures.

Example 13 Evaluation of an Exemplary Polypeptide Provided in Accordance with Aspects of the Present Disclosure in a Cerulean-Induced In Vivo Mouse Model for Acute Pancreatitis

Hyperstimulation acute pancreatitis is induced by either 7 or 12 intraperitoneal injections of 50 mg/kg caerulein hourly, with saline controls. Mice receive seven intraperitoneal injections of either 10, 50, 100 or 250 ng/kg of H_(C)/A peptide, beginning 2 hours after the first caerulein injection. Mice are sacrificed 12 hours after disease induction and assessed for serum amylase, pancreatic oedema, pancreatic trypsin activity, pancreatic myeloperoxidase activity, and serum interleukin (IL-6) (normalized to untreated caerulein group).

Results: Dose-dependent protective effects of H_(C)/A peptide on the severity of caerulein acute pancreatitis are observed at 12 hours. This improvement is consistent with experimental data described in Examples 1, 3, 5, 6 and 7, wherein fibroblasts treated with a polypeptide corresponding to the binding domain of BoNT/A (H_(C)/A) were shown to have reduced expression of genes associated with myofibroblast formation, including S100A4 and ACTA2, and modulated expression and secretion of extracellular matrix (ECM) proteins and proteinases, including fibronectin, collagen and MMP1, that function to properly maintain, renew and repair ECM tissue structures.

Example 14 Use of an Exemplary Polypeptide Provided in Accordance with Aspects of the Present Disclosure to Prevent Intra-Abdominal Adhesions

Postoperative intraperitoneal adhesions (PIAs) are one of the most important problems surgeons have to face after laparotomies. In this study, we aim to evaluate the effectiveness of local application of H_(C)/A peptide in various dosages on the prevention of intra-abdominal adhesions in rats with experimental intra-abdominal adhesions.

Forty Wistar Albino female rats are randomly separated into 4 groups. The 4 groups are determined as follows: Control (group 1, n=10); Sham (group 2, n=10); 10-mg/kg low-dose H_(C)/A peptide (group 3, n=10) and 30-mg/kg high dose HC/A peptide (group 4, n=10). Subserosal injuries are created on the caecum of all rats. Laparotomy is performed on the fifth day. Adhesion scores, histopathological examination, and E-cadherin expression levels are evaluated.

Results: General adhesion scores for groups 1 and 2 (Control and Sham) are determined to be significantly high when compared to group 4 (high dose H_(C)/A peptide) (P<0.001). A significant difference is also determined between groups 3 and 4 (low versus high dose H_(C)/A peptide) in terms of general adhesion scores (P<0.05). In pair comparisons, a significant decrease in the high dose H_(C)/A peptide group (group 4) when compared to groups 1 and 2 (Control and Sham) in terms of neovascularization, fibroblast density, collagen deposition and inflammatory cell count is determined (P<0.05).

Conclusion: In this study, high-dose H_(C)/A peptide is determined to be an effective agent in preventing postoperative PIAs. This improvement is consistent with experimental data described in Examples 1, 3, 5, 6 and 7, wherein fibroblasts treated with a polypeptide corresponding to the binding domain of BoNT/A (H_(C)/A) were shown to have reduced expression of genes associated with myofibroblast formation, including S100A4 and ACTA2, and modulated expression and secretion of extracellular matrix (ECM) proteins and proteinases, including fibronectin, collagen and MMP1, that function to properly maintain, renew and repair ECM tissue structures. It is also consistent with Examples 4 showing that the binding domain of BoNT/A (H_(C)/A) reduces fibroblast contractility.

Example 15 Use of an Exemplary Polypeptide Provided in Accordance with Aspects of the Present Disclosure in a Rat Model to Promote Scarless Wound Healing

Wounds are created in six anesthetized CD hairless female rats ranging 260 g-310 g. Sterile biopsy punches are used to create two symmetrical full-thickness excisional wounds beside the midline with an average diameter of 11.5 mm.

H_(C)/A peptide is dissolved in sterile phosphate-buffered saline to a final concentration 5, 10 and 20 pM and injected intradermally with 25 μL per injection site at 7-9 sites surrounding the excision edge. For comparison, injection with sterile phosphate-buffered saline is done in parallel.

Photos and measurements of individual wounds are taken at day 0, 3, 7, 10, 14, and 21. Tissue biopsy samples are collected on day 21 to access collagen structure by histology.

Results: Initially, day 3-9, the H_(C)/A peptide treated wounds heal slower. However, on day 21 the scars are shorter, flatter, and less visible in the HC/A peptide treated groups, and the collagen fibers appear more organized.

Conclusion: The results suggest that injection with H_(C)/A peptide immediately post-operatively results in wound healing with less scarring. This improvement is consistent with experimental data described in Examples 1, 3, 5, 6 and 7, wherein fibroblasts treated with a polypeptide corresponding to the binding domain of BoNT/A (H_(C)/A) were shown to have reduced expression of genes associated with myofibroblast formation, including S100A4 and ACTA2, and modulated expression and secretion of extracellular matrix (ECM) proteins and proteinases, including fibronectin, collagen and MMP1, that function to properly maintain, renew and repair ECM tissue structures. It is also consistent with Examples 4 showing that the binding domain of BoNT/A (H_(C)/A) reduces fibroblast contractility.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described in the foregoing paragraphs. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. In case of conflict, the present specification, including definitions, will control.

All United States patents and published or unpublished United States patent applications cited herein are incorporated by reference. All published foreign patents and patent applications cited herein are hereby incorporated by reference. All published references, documents, manuscripts, scientific literature cited herein are hereby incorporated by reference. All identifier and accession numbers pertaining to scientific databases referenced herein (e.g., PUBMED, NCBI, GENBANK, EBI) are hereby incorporated by reference. 

1. A method for treating and/or reducing the occurrence of an extracellular matrix (ECM) disease, connective tissue disease, endothelial disease or a fibrotic disorder in a subject in need thereof, comprising: administering to the subject a composition comprising an effective amount of a polypeptide having an amino acid sequence with at least about 90% sequence identity to a binding domain of a botulinum toxin, wherein the molecular weight of the polypeptide is between about 4 kDa to about 60 kDa, and wherein treating and/or reducing the occurrence of an ECM disease, connective tissue disease, endothelial disease or a fibrotic disorder does not involve paralysis of a target muscle.
 2. The method of claim 1, wherein the fibrotic disorder is selected from the group consisting of, dermal scars, wound scars, surgical scars, postoperative intraperitoneal adhesions, fibroma, liver fibrosis, cirrhosis, pancreatitis, benign prostatic hyperplasia, fibrotic bladder, interstitial cystitis, pulmonary fibrosis, kidney fibrosis, glomerulosclerosis, atrial fibrosis, endomyocardial fibrosis, glial scar, arterial stiffness, arthrofibrosis, cystic fibrosis, non-cystic fibrosis, crohn's disease, dupuytren's contracture, mediastinal fibrosis, myelofibrosis, peyronie's disease, nephrogenic systemic fibrosis, progressive massive fibrosis, retroperitoneal fibrosis, adhesive capsulitis, mixed connective tissue disease, ocular fibrosis, osteoarthritis, scleroderma, and asthma.
 3. The method of claim 1, wherein the fibrotic disorder is selected from the group consisting of fibrotic breast disease; keloid scar; liver fibrosis; fibrotic bladder; hyperplasia of fibrous tissue; pancreatitis; adhesion; scarring; or combinations thereof.
 4. The method of claim 1, wherein the subject is a human subject.
 5. The method of claim 1, wherein the molecular weight of the polypeptide is between about 10 kDa to about 60 kD.
 6. The method of claim 1, wherein the polypeptide comprises an amino acid sequence with at least about 95% sequence identity to a binding domain of a botulinum toxin.
 7. The method of claim 1, wherein the botulinum toxin is selected from the group consisting of Botulinum toxin serotype A (BoNT/A), Botulinum toxin serotype B (BoNT/B), Botulinum toxin serotype C₁ (BoNT/C₁), Botulinum toxin serotype D (BoNT/D), Botulinum toxin serotype E (BoNT/E), Botulinum toxin serotype F (BoNT/F), Botulinum toxin serotype F (BoNT/FA), Botulinum toxin serotype G (BoNT/G), Botulinum toxin serotype H (BoNT/H), Botulinum D/C mosaic (BoNT/DC), Botulinum C/D mosaic (BoNT/CD), Botulinum toxin serotype X (BoNT/X), and Enterococcus sp. BoNT J (eBoNT/J).
 8. The method of claim 1, wherein the polypeptide comprises an amino acid sequence having at least 30% homology to a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 19, SEQ ID NOs: 3-5, SEQ ID NO: 6, SEQ ID NO: 20, SEQ ID Nos: 7-10, SEQ ID NO: 11, SEQ ID NO: 21, SEQ ID NOs: 12-18 and SEQ ID NOs: 25-27.
 9. The method of claim 1, wherein the polypeptide comprises, consists essentially of, or consists of a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 19, SEQ ID NOs: 3-5, SEQ ID NO: 6, SEQ ID NO: 20, SEQ ID Nos: 7-10, SEQ ID NO: 11, SEQ ID NO: 21, SEQ ID NOs: 12-18 and SEQ ID NOs: 25-27. 